Genes encoding proteins that interact with the tub protein

ABSTRACT

The present invention relates to the discovery of novel genes encoding Tub interactor (TI) polypeptides. Therapeutics, diagnostics and screening assays based on these molecules are also disclosed.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/715,032,filed Sep. 17, 1996, now abandoned, the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Obesity represents the most prevalent of body weight disorders, and itis the most important nutritional disorder in the western world, withestimates of its prevalence ranging from 30% to 50% within themiddle-aged population. Other body weight disorders, such as anorexianervosa and bulimia nervosa which together affect approximately 0.2% ofthe female population of the western world, also pose serious healththreats. Further, such disorders as anorexia and cachexia (wasting) arealso prominent features of other diseases such as cancer, cysticfibrosis, and AIDS.

Obesity, defined as an excess of body fat relative to lean body mass,also contributes to other diseases. For example, this disorder isresponsible for increased incidences of diseases such as coronary arterydisease, hypertension, stroke diabetes, hyperlipidemia and some cancers.(See, e.g., Nishina, P. M. et al. (1994) Metab. 43:554-558; Grundy, S.M. and Barnett, J. P. (1990) Dis. Mon. 36:641-731) Obesity is not merelya behavioral problem, i.e., the result of voluntary hyperphagia. Rather,the differential body composition observed between obese and normalsubjects results from differences in both metabolism andneurologic/metabolic interactions. These differences seem to be, to someextent, due to differences in gene expression, and/or level of geneproducts or activity (Friedman, J. M. et al. (1991) Mammalian Gene1:130-144).

The epidemiology of obesity strongly shows that the disorder exhibitsinherited characteristics (Stunkard (1990) N. Eng. J. Med. 322:1483).Moll et al. have reported that, in many populations, obesity seems to becontrolled by a few genetic loci (Moll et al. (1991) Am. J. Hum. Gen.49:1243). In addition, human twin studies strongly suggest a substantialgenetic basis in the control of body weight, with estimates ofheritability of 80-90% (Simopoulos, A. P. and Childs B., eds., 1989, in"Genetic Variation and Nutrition in Obesity", World Review of Nutritionand Diabetes 63, S. Karger, Basel, Switzerland; Borjeson, M., 1976,Acta. Paediatr. Scand. 65:279-287).

Studies of non-obese persons who deliberately attempted to gain weightby systematically over-eating were found to be more resistant to suchweight gain and able to maintain an elevated weight only by very highcaloric intake. In contrast, spontaneously obese individuals are able tomaintain their status with normal or only moderately elevated caloricintake. In addition, it is a commonplace experience in animal husbandrythat different strains of swine, cattle, etc., have differentpredispositions to obesity. Studies of the genetics of human obesity andof models of animal obesity demonstrate that obesity results fromcomplex defective regulation of both food intake, food induced energyexpenditure and of the balance between lipid and lean body anabolism.

There are a number of genetic diseases in man and other species whichfeature obesity among their more prominent symptoms, along with,frequently, dysmorphic features and mental retardation. For example,Prader-Willi syndrome (PWS; reviewed in Knoll, J. H. et al. (1993) Am.J. Med. Genet. 46:2-6) affects approximately 1 in 20,000 live births,and involves poor neonatal muscle tone, facial and genital deformities,and generally obesity.

In addition to PWS, many other pleiotropic syndromes which includeobesity as a symptom have been characterized (e.g. Ahlstroem, Carpenter,Bardet-Biedl, Cohen, and Morgagni-Stewart-Monel Syndromes). Thesesyndromes are more genetically straightforward and appear to involveautosonial recessive alleles.

A number of models exist for the study of obesity (see, e.g., Bray, G.A. (1992) Prog. Brain Res. 93:333-341, and Bray, G. A. (1989) Amer. J.Clin. Nutr. 5:891-902). For example, animals having mutations which leadto syndromes that include obesity symptoms have also been identified.Attempts have been made to utilize such animals as models for the studyof obesity, and the best studied animal models, to date, for geneticobesity are mice. For reviews, see e.g., Friedman, J. M. et al. (1991)Mamm. Gen. 1:130-144; Friedman, J. M. and Liebel, R. L. (1992) Cell69:217-220.

Studies utilizing mice have confirmed that obesity is a very complextrait with a high degree of heritability. Mutations at a number of locihave been identified which lead to obese phenotypes. These include theautosomal recessive mutations obese (ob), diabetes (db), fat (fat) andtubby (tub). In addition, the autosomal dominant mutations Yellow at theagouti locus and Adipose (Ad) have been shown to contribute to an obesephenotype.

The ob and db mutations are on chromosomes 6 and 4, respectively, butlead to clinically similar pictures of obesity, evident starting atabout one month of age, which include hyperphagia, severe abnormalitiesin glucose and insulin metabolism, very poor thermoregulation andnon-shivering thermogenesis, and extreme torpor and underdevelopment ofthe lean body mass.

The ob gene and its human homologue have recently been cloned (Zhang, Y.et al., (1994) Nature 372:425-432). The gene appears to produce a 4.5 kbadipose tissue messenger RNA which contains a 167 amino acid openreading frame. The predicted amino acid sequence of the ob gene productindicates that it is a secreted protein and may, therefore, play a roleas part of a signaling pathway from adipose tissue which may serve toregulate some aspect of body fat deposition.

The db locus encodes a high affinity receptor for the ob gene product(Chen, H. et al. Cell 84:491-495). The db gene product is a singlemembrane-spanning receptor most closely related to the gp 130 cytokinereceptor signal transducing component (Tartaglia, L. A. et al. (1995)Cell 83:1263-1271).

Homozygous mutations at either the fat or tub loci cause obesity whichdevelops more slowly than that observed in ob and db mice (Coleman, D.L., and Eicher, E. M. (1990) J. Heredity 81:424-427), with tub obesitydeveloping slower than that observed in fat animals. This feature of thetub obese phenotype makes the development of tub obese phenotype closestin resemblance to the manner in which obesity develops in humans. Evenso, however, the obese phenotype within such animals can becharacterized as massive in that animals eventually attain body weightswhich are nearly two times the average weight seen in normal mice.tub/tub mice develop insulin resistance with their weight gain but donot progress to overt diabetes.

In addition to obesity, retinal defects, hearing loss and infertilityhave all been observed in tub mice (Heckenlively, 1988, in RetinitisPigmentosa, Heckenlively, ed., Lippincott, Philadelphia, pp. 221-235;Coleman, D. L. & Eicher, E. M., 1990, J. Hered. 81 :424-4a27;Ohlemiller, K. K. et al. (1995) Aeuroreport 6:845-849). Several humansyndromes exist in which such defects are found to co-exist with anobesity phenotype, including Bardet-Biedl syndrome, Ahlstroem syndrome,polycystic ovarian disease and Usher's syndrome.

The fat mutation has been mapped to mouse chromosome 8, while the tubmutation has been mapped to mouse chromosome 7. According to Naggert etal., the fat mutation has recently been identified (Naggert, J. K., etal. (1995) Nature Genetics 10:135-141). Specifically, the fat mutationappears to be a mutation within the Cpe locus, which encodes thecarboxypeptidase (Cpe) E protein. Cpe is an exopeptidase involved in theprocessing of prohormones, including proinsulin.

The dominant Yellow mutation at the agouti locus, causes a pleiotropicsyndrome which causes moderate adult onset obesity, a yellow coat color,and a high incidence of tumor formation (Herberg, L. and Coleman, D. L.(1977) Metabolism 26:59), and an abnormal anatomic distribution of bodyfat (Coleman, D. L. (1978) Diabetologia 14:141-148). This mutation mayrepresent the only known example of a pleiotropic mutation that causesan increase, rather than a decrease, in body size. The mutation causesthe widespread expression of a protein which is normally seen only inneonatal skin (Michaud, E. J. et al. (1994) Genes Devel. 8:1463-1472).

Other animal models include fa/fa (fatty) rats, which bear manysimilarities to the ob/ob and db/db mice, discussed above. Onedifference is that, while fa/fa rats are very sensitive to cold, theircapacity for non-shivering thermogenesis is normal. Torpor seems to playa larger part in the maintenance of obesity in fa/fa rats than in themice mutants. In addition, inbred mouse strains such as NZO mice andJapanese KK mice are moderately obese. Certain hybrid mice, such as theWellesley mouse, become spontaneously fat. Further, several desertrodents, such as the spiny mouse, do not become obese in their naturalhabitats, but do become so when fed on standard laboratory feed.

Animals which have been used as models for obesity have also beendeveloped via physical or pharmacological methods. For example,bilateral lesions in the vetromedial hypothalamus (VMH) andventrolateral hypothalamus (VLH) in the rat are associated,respectively, with hyperphagia and gross obesity and with aphagia,cachexia and anorexia. Further, it has been demonstrated that feedingmonosodiumglutamate (MSG) or gold thioglucose to newborn mice alsoresults in an obesity syndrome.

In summary, therefore, obesity, which poses a major, worldwide healthproblem, represents a highly heritable trait. Given the severity,prevalence and potential heterogeneity of such disorders, there exists agreat need for the identification genes involved in the control of bodyweight.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of novel molecules,referred to herein as "Tub Interactor" ("TI") nucleic acid andpolypeptide molecules. Exemplary novel TI molecules are contained in andencoded by: 1) E. coli plasmid ptyhq049, which was deposited with theAmerican Type Culture Ccllection (ATCC) on Aug. 6, 1996 and has beenassigned ATCC designation number 98125; 2 E. coli plasmid ptyhq054,which was deposited with the American Type Culture Collection (ATCC) onAug. 6, 1996 and has been assigned ATCC designation number 98126; 3) E.coli plasmid ptyhq058, which was deposited with the American TypeCulture Collection (ATCC) on Aug. 6, 1996 and has been assigned ATCCdesignation number 98127; and 4) E. coli plasmid ptyhq036, which wasdeposited with the American Type Culture Collection (ATCC) on Aug. 6,1996 and has been assigned ATCC designation number 98128.

Six novel TI genes were cloned and identified based on their ability tointeract with the C-terminus (i.e. the last 44 amino acids) of htub in atwo hybrid assay as further described in the following Examples. hTI-1(FIG. 1 (SEQ ID NO:1)) is a 1386 base pair nucleic acid encoding aserine protease. hTI-2 (FIG. 2 (SEQ ID NO:2)) is a 2103 base pairnucleic acid containing ANK (i.e. ankyrin) repeats. hTI-3 (FIG. 3 (SEQID NO:3)) is a 1048 base pair nucleic acid containing TPR repeats (i.e.tetraticopeptide repeats) and also DNAJ repeats. mTI-3 (FIG. 4 (SEQ IDNO:4)) is a 1700 base pair nucleic acid that is the murine homologue ofhTI-3. hTI-4 (FIG. 5 (SEQ ID NO:5)) is a 1421 base pair nucleic acidthat contains RING finger repeat,; and also Zinc finger repeats. mTI-4(FIG. 6 (SEQ ID NO:6)) is a 2121 base pair nucleic acid that is themurine homologue of hTI-4. A final TI gene (hTI-5) was identified asencoding human serine palmitoyltransferase (GenBank Accession No.U15555).

In one aspect, the invention features isolated vertebrate TI nucleicacid molecules. The disclosed molecules can be non-coding, (e.g. probe,antisense or ribozyme molecules) or can encode a functional TIpolypeptide (e.g. a polypeptide which specifically modulates, e.g., byacting as either an agonist or antagonist, at least one bioactivity ofthe human TI polypeptide). In one embodiment, the nucleic acid moleculescan hybridize to the TI gene contained in any of ATCC designationnumbers 98125, 98126, 981257, or 98128 or to the complement of the TIgene contained in any of ATCC designation numbers 98125, 98126, 981257,or 98128. In another embodiment, the nucleic acids of the presentinvention can hybridize to a vertebrate TI gene or to the complement ofa vertebrate TI gene. In a further embodiment, the claimed nucleic acidcan hybridize with the nucleic acid sequence, designated in SEQ IDNOs:1-6 or to the complement to the nucleic acid sequence designated inSEQ ID NOs:1-6. In a preferred embodiment, the hybridization isconducted under mildly stringent or stringent conditions.

In further embodiments, the nucleic acid molecule is a TI nucleic acidthat is at least 70%, preferably 80%, more preferably 85%, and even morepreferably at least 90% or 95% homologous in sequence to any of thenucleic acids shown as SEQ ID NOs:1-6 or to the complement of thenucleic acid shown as SEQ ID NOs:1-6. In a further embodiment, thenucleic acid molecule is a TI nucleic acid that is at least 70%,preferably 80%, more preferably 85% and even more preferably at least90% or 95% similar in sequence to the TI gene contained in any of ATCCdesignation numbers 98125, 98126, 981257, or 98128 or to the complementof the TI gene contained in any of ATCC designation numbers 98125,98126, 981257, or 98128.

The invention also provides probes and primers comprising substantiallypurified oligonucleotides, which correspond to a region of nucleotidesequence which hybridizes to at least 6 consecutive nucleotides of anyof the sequences set forth as SEQ ID NOs:1-6 or complements of any ofthe sequences set forth as SEQ ID NOs:1-6 or naturally occurring mutantsthereof. In preferred embodiments, the probe/primer further includes alabel group attached thereto, which is capable of being detected.

For expression, the subject nucleic acids can include a transcriptionalregulatory sequence, e.g. at least one of a transcriptional promoter(e.g., for constitutive expression or inducible expression) ortranscriptional enhancer sequence, which regulatory sequence is operablylinked to the gene sequence. Such regulatory sequences in conjunctionwith a TI nucleic acid molecule can provide a useful vector for geneexpression. This invention also describes host cells transfected withsaid expression vector whether prokaryotic or eukaryotic and in vitro(e.g. cell culture) and in vivo (e.g. transgenic) methods for producingTI proteins by employing said expression vectors.

In another aspect, the invention features isolated TI polypeptides,preferably substantially pure preparations e.g. of plasma purified orrecombinantly produced polypeptides. In preferred embodiments, thepolypeptide is able to bind to the C-terminus (e.g. the last 44 aminoacids) of the human tub protein. In particularly preferred embodiments,the subject polypeptides, whether agonists or antagonists, can suppressthe development and/or progression of a weight disorder (obesity,cachexia or anorexia nervosa) or a related disorder (e.g. diabetes).

In a preferred embodiment, the TI polypeptide is encoded by a nucleicacid which hybridizes with any of the nucleic acid sequences representedin SEQ ID NOs:1-6 or with the gene or gene fragment contained in any ofATCC Designation Nos. designation numbers 98125, 98126, 981257, or98128. The subject TI proteins also include modified protein, which areresistant to post-translationil modification, as for example, due tomutations which alter modification sites (such as tyrosine, threonine,serine or aspargine residues), or which prevent glycosylation of theprotein, or which prevent interaction of the protein with intracellularproteins involved in signal transduction.

The TI polypeptides can comprise a full length protein or it cancomprise a fragment corresponding to one or more particularmotifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50,100, 150 or 200 amino acids in length. In preferred embodiments, thepolypeptide includes a sufficient portion of the domain that interactswith the C-terminus (i.e. the last 44 amino acids) of normal human tub.

Another aspect of the invention features chimeric molecules (e.g. fusionproteins) comprised of a TI protein. For instance, the TI protein can beprovided as a recombinant fusion protein which includes a secondpolypeptide portion, e.g., a second polypeptide having an amino acidsequence unrelated (heterologous) to the TI polypeptide, (e.g. thesecond polypeptide portion is glutathione-S-transferase, an enzymaticactivity such as alkaline phosphatase or an epitope tag).

Yet another aspect of the present invertion concerns an immunogencomprising a TI polypeptide in an immunogenic preparation, the immunogenbeing capable of eliciting an immune response specific for a TIpolypeptide; e.g. a humoral response, an antibody response and/orcellular response. In a preferred embodiment, the immunogen comprises anantigenic determinant, e.g. a unique determinant of a protein encoded byany of the nucleic acids SEQ ID NOs:1-6.

A still further aspect of the present invention features antibodies andantibody preparations specifically reactive with an epitope of a TIprotein.

The invention also features transgenic non-human animals which include(and preferably express) a heterologous form of a TI gene describedherein, or which misexpress an endogenous TI gene (e.g., an animal inwhich expression of one or more of the subject TI proteins isdisrupted). Such a transgenic animal can serve as an animal model forstudying cellular and tissue disorders comprising mutated ormis-expressed TI alleles or for use in drug screening. Alternatively,such a trlnsgenic animal can be useful for expressing recombinant TIpolypeptides.

In yet another aspect, the invention provides assays, e.g., forscreening test compounds to identify modulators (e.g., inhibitors, oralternatively, potentiators) of an interaction between a TI protein and,for example, a tub polypeptide. An exemplary method includes the stepsof (i) combining a TI protein or bioactive fragment thereof, a TIprotein target molecule (such as Tub), and a test compound, e.g., underconditions wherein, but for the test compound, the TI protein and targetmolecule are able to interact; and (ii) detecting the formation of acomplex which includes the TI protein and the target polypeptide eitherby directly quantitating the complex, by measuring inductive effects ofthe TI protein, or, in the instance of a substrate, measuring theconversion to product. A statistically significant change, such as adecrease, in the interaction of the TI protein and target molecule inthe presence of a test compound (relative to what is detected in theabsence of the test compound) is indicative of a modulation (e.g.,inhibition or potentiation of the interaction between the TI protein andthe target molecule).

Yet another aspect of the present invention concerns a method formodulating apoptosis in a cell by modulating TI bioactivity, (e.g., bypotentiating or disrupting certain protein-protein interactions). Ingeneral, whether carried out in vivo, in vitro, or in situ, the methodcomprises treating the cell with an effective amount of a TI therapeuticso as to alter, relative to the cell in the absence of treatment, lipiduptake by the cell. Accordingly, the method can be carried out with TImodulating agents such as peptide and peptidomimetics or other moleculesidentified in the above-referenced drug screens which agonize orantagonize the effects of signaling in a biochemical pathway involving aTI protein. Other modulating agents for use as therapeutics includeantisense constructs for inhibiting expression of TI proteins, anddominant negative mutants of TI proteins which competitively inhibitligand interactions upstream and signal transduction downstream of thewild-type TI protein.

A further aspect of the present invention provides a method ofdetermining if a subject is at risk for a disorder characterized byinappropriate TI protein expression, such as, for example, a weightdisorder (e.g. obesity, cachexia or anorexia nervosa) or a relateddisorder, such as diabetes. The method includes detecting, in a tissueof the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding a TIprotein, e.g. represented in any of SEQ ID NOs:1-6 or a homologuethereof; or (ii) the mis-expression of a TI gene. In preferredembodiments, detecting the genetic lesion includes ascertaining theexistence of at least one of: a deletion of one or more nucleotides froma TI gene; an addition of one or more nucleotides to the gene, asubstitution of one or more nucleotides of the gene, a gross chromosomalrearrangement of the gene; an alteration in the level of a messenger RNAtranscript of the gene; the presence of a non-wild type splicing patternof a messenger RNA transcript of the gene; a non-wild type level of theprotein; and/or an aberrant level of soluble TI protein.

For example, detecting the genetic lesion can include (i) providing aprobe/primer comprised of an oligonucleotide which hybridizes to a senseor antisense sequence of a TI gene or naturally occurring mutantsthereof, or 5' or 3' flanking sequences naturally associated with the TIgene; (ii) contacting the probe/primer to an appropriate nucleic acidcontaining sample; and (iii) detecting, by hybridization of theprobe/primer to the nucleic acid, the presence or absence of the geneticlesion; e.g. wherein detecting the lesion comprises utilizing theprobe/primer to determine the nucleotide sequence of the TI gene and,optionally, of the flanking nucleic acid sequences. For instance, theprimer can be employed in a polymerase chain reaction (PCR) or in aligation chain reaction (LCR). In alternate embodiments, the level of aTI prote in is detected in an immunoassay using an antibody which isspecifically immunoreactive with the TI protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence of a novel human TI gene, E. coli plasmidptyhq049, ATCC designation no. 98125 (hTI-1) (SEQ ID NO:1) and a deducedamino acid sequence.

FIG. 2 shows the DNA sequence of a novel human TI gene, E. coli plasmidptyhq058, ATCC designation no. 98127 (hTI-2) (SEQ ID NO:2) and a deducedamino acid sequence.

FIG. 3 shows the DNA sequence of a novel human TI gene, E. coli plasmidptyhq036, ATCC designation no. 98128 (hTI-3) (SEQ ID NO:3) and a deducedamino acid sequence.

FIG. 4 shows the DNA sequence of a novel murine TI gene, E. coli plasmidptyht101 (mTI-3) (SEQ ID NO:4) and a deduced amino acid sequence.

FIG. 5 shows the DNA sequence of a novel human TI gene, E. coli plasmidptyhq054, ATCC designation no. 98126 (hTI-4) (SEQ ID NO:5) and a deducedamino acid sequence.

FIG. 6 shows the DNA sequence of a novel murine TI gene, E. coli plasmidptyht102 (mTI-4) (SEQ ID NO:6) and a deduced amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel genes, referredto herein as the "Tub interactor" or "TI" genes, which function inbiochemical pathways involved in weight control and/or related disorder,such as diabetes.

Six novel TI genes were cloned and identified based on their ability tointeract with the C-terminus (i.e. the last 44 amino acids) of htub in atwo hybrid assay as further described in the following Examples. hTI-1is a 1386 base pair nucleic acid, the sequence of which is presented inFIG. 1 (SEQ ID NO:1). Based on sequence analysis, the polypeptideencoded by the gene is a putative serine protease.

hTI-2 is a 2103 base pair nucleic acid, the sequence of which ispresented in FIG. 2 (SEQ ID NO:2). The sequence contains ANK (i.e.ankyrin) repeats indicating that the protein encoded by the nucleic acidspecifically recognize proteins and/or nucleic acid molecules (Michaely,P. and V. Bennett (1992) Trends in Cell Biology 2:127-129). Based onNorthern analysis, a major band of 2.4 kb and a minor band of 8 kbcorresponding to TI-2 was expressed in all human tissue and cell linestested. However, the highest expression occurred in the testis,pancreas, liver, uterus and brain.

hTI-3 is a 1048 base pair nucleic acid, the sequence of which ispresented in FIG. 3 (SEQ ID NO:3). The sequence contains TPR repeats(i.e. tetraticopeptide repeats) and also DNAJ repeats, indicating thatthe protein encoded by the nucleic acid is involved in protein-proteininteractions. Based on Northern analysis, a major band of 2.2 kb and aminor band of 1.2 kb corresponding to hTI-3 was expressed in all humantissue and cell lines tested. However, the highest expression occurredin skeletal muscle, liver, heart and testis.

mTI-3 is a 1700 base pair nucleic acid, the sequence of which ispresented in FIG. 4 (SEQ ID NO:4). A sequence comparison of a 1035 baseregion indicates that the human and mouse genes are 86.8% identical.Like the human, the murine sequence contains TPR repeats (i.e.tetraticopeptide repeats) (Silkorski, R. J. et al., (Jan. 26, 1990) Cell60:307-317; Lee, T. G. et al. (April 1994) Mol. Cell. Biol.14:2331-2342; Barber, G. N et al. (May 1994) Proc. Natl. Acad. Sci. USA91:4278-4282) and also DNAJ repeats (Silver, P. A. (Jul. 16, 1993) Cell74:5-6), indicating that the protein encoded by the nucleic acid isinvolved in protein-protein interactions. Based on Northern analysis, amajor band of 1.4 kb corresponding to mTI-3 was expressed in all murinetissue tested (both tub and B6). However, the highest expressionoccurred in skeletal muscle, liver, heart and testis.

hTI-4 is a 1421 base pair nucleic acid, the sequence of which ispresented in FIG. 5 (SEQ ID NO:5). The sequence contains RING fingerrepeats (Saurin, A. J. et al. (June 1996) TIBS 21:) and also Zinc fingerrepeats (Lovering R. et al. (March 1993) Proc. Natl. Acad. Sci. USA90:2112-2116) indicating that the protein encoded by the nucleic acid isinvolved in nucleic acid (i.e. DNA or RNA) interactions. Based onNorthern analysis, bands of 4 and 3 kb corresponding to hTI-4 wasexpressed in all human tissue and cell lines tested. In addition, a 1.4kb band was strongly expressed in testis. Further, a band correspondingto 2.4 kb was expressed in the human SHEP, SHSY5Y, SKNMC and SKNSH celllines.

mTI-4 is a 2121 base pair nucleic acid, the sequence of which ispresented in FIG. 6 (SEQ ID NO:6). A sequence comparison of a 959 baseregion indicates that the human and mouse genes are 86.8% identical.Like the human, the murine sequence contains RING finger repeats andalso Zinc finger repeats indicating that the protein encoded by thenucleic acid is involved in nucleic acid (i.e. DNA or RNA) interactions.Based on Northern analysis, major bands of 3.0 and 2.4 kb correspondingto mTI-4 was expressed in all murine tissue tested. In addition, a 1.4kb band was expressed in Tub and B6 mouse.

Another TI gene (hTI-5) was identified as encoding human serinepalmitoyltransferase (GenBank Accession No. U15555) , an enzyme thatcatalyzes the committed step in sphingolipid and ceramide biosythesis.Ceramide is a second messenger that regulates apoptosis via PP2A(Nickels, J. T. and J. R. Broach (1996) Genes & Development 10:382-394.

The cDNAs corresponding to TI gene transcripts were initially clonedfrom human breast tissue based on the ability of their encoded proteinsto bind to the C-terminal domain (i.e. the last 44 amino acids) of thehtub gene product in an assay that detects protein/protein interactions,placing the TI gene products in the same biochemical pathway as tub. Thetub protein is described in U.S. patent application Ser. No. 08/631,200filed on Apr. 12, 1996.

Accordingly, certain aspects of the present invention relate to nucleicacid molecules encoding TI proteins, the TI proteins, antibodiesimmunoreactive with TI proteins, and preparations of such compositions.in addition, drug discovery assays are provided for identifying agentswhich can modulate the biological function of TI proteins, such as byaltering the interaction of TI molecules with either downstream orupstream elements in the tub signal transduction pathway. Such agentscan be useful therapeutically, for example, to modulate weight controland/or diabetes. Moreover, the present invention provides diagnostic andtherapeutic assays and reagents for detecting and treating disordersinvolving, for example, aberrant expression (or loss thereof) of TIgenes. Other aspects of the invention are described below or will beapparent to those skilled in the art in light of the present disclosure.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

"Cells," "host cells" or "recombinant host cells" are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A "chimeric protein" or "fusion protein" is a fusion of a first aminoacid sequence encoding one of the subject TI polypeptides with a secondamino acid sequence defining a domain (e.g. polypeptide portion) foreignto and not substantially homologous with any domain of one of the TIpolypeptides. A chimeric protein may present a foreign domain which isfound (albeit in a different protein) in an organism which alsoexpresses the first protein, or it may be an "interspecies","intergenic", etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula X-TI-Y, wherein TI represents a portion of theprotein which is derived from one of the TI proteins, and X and Y areindependently absent or represent amino acid sequences which are notrelated to one of the TI amino acid sequences in an organism, includingnaturally occurring mutants.

"Complementary" sequences as used herein refer to sequences which havesufficient complementarity to bc ablc to hybridize, forming a stableduplex.

A "delivery complex" shall mean a targeting means (e.g. a molecule thatresults in higher affinity binding of a gene, protein, polypeptide orpeptide to a target cell surface and/or increased cellular uptake by atarget cell). Examples of targeting means include: sterols (e.g.cholesterol), lipids (e.g. a cationic lipid, virosome or liposome),viruses (e.g. adenovirus, adeno-associated virus, and retrovirus) ortarget cell specific binding agents (e.g. ligands recognized by targetcell specific receptors). Preferred complexes are sufficiently stable invivo to prevent significant uncoupling prior to internalization by thetarget cell. However, the complex is cleavable under appropriateconditions within the cell so that the gene, protein, polypeptide orpeptide is released in a functional form.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term "DNAsequence encoding a TI polypeptide" may thus refer to one or more geneswithin a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indiffecrcnccs in amino acid sequence of the encoded polypeptide yet stillencode a protein with the same biological activity.

As used herein, the term "gene" or "recombinant gene" refers to anucleic acid molecule comprising an open reading frame encoding one ofthe TI polypeptides of the present invention, including both exon and(optionally) intron sequences. A "recombinant gene" refers to nucleicacid molecule encoding a TI polypeptide and comprising TIprotein-encoding exon sequences, though it may optionally include intronsequences which are either derived from a chromosomal TI gene or from anunrelated chromosomal gene. Exemplary recombinant genes encoding thesubject TI polypeptides are represented in the appended SequenceListing. The term "intron" refers to a DNA sequence present in a givengene which is not translated into protein and is generally found betweenexons.

"Homology" or "identity" or "similarity" refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An "unrelated" or "non-homologous" sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the TI sequences of the present invention.

The term "interact" as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a yeast two hybrid assay. The term interact is also meant toinclude "binding" interactions between molecules. Interactions may beprotein-protein or protein-nucleic acid in nature.

The term "isolated" as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject TI polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks the TIgene in genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an "isolated nucleicacid" is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm "isolated" is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

The term "modulation" as used herein refers to both upregulation, i.e.,stimulation, and downregulation, i.e. suppression, of a response.

The "non-human animals" of the invention include mammalians such asrodents, non-human primates, sheep, dog, cow, chickers, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse. The term"chimeric animal" is used herein to refer to animals in which therecombinant gene is found, or in which the recombinant is expressed insome but not all cells of the animal. The term "tissue-specific chimericanimal" indicates that one of the recombinant TI genes is present and/orexpressed or disrupted in some tissues but not others.

As used herein, the term "nucleic acid" refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used herein, the term "promoter" means a DNA sequence that regulatesexpression of a selected DNA sequence operably linked to the promoter,and which effects expression of the selected DNA sequence in cells. Theterm encompasses "tissue specific" promoters, i.e. promoters, whicheffect expression of the selected DNA sequence only in specific cells(e.g. cells of a specific tissue). The term also covers so-called"leaky" promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

The terms "protein", "polypeptide" and "peptide" are used interchangablyherein when referring to a gene product.

The term "recombinant protein" refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a TI polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. Moreover, the phrase "derived from",with respect to a recombinant TI gene, is meant to include within themeaning of "recombinant protein" those proteins having an amino acidsequence of a native TI protein, or an amino acid sequence similarthereto which is generated by mutations including substitutions anddeletions (including truncation) of a naturally occurring form of theprotein.

As used herein, the term "specifically hybridizes" or "specificallydetects" refers to the ability of a nucleic acid molecule of theinvention to hybridize to at least approximately 6, 12, 20, 30, 50, 100,150, 200, 300, 350, 400, or 425 consecutive nucleotides of a vertebrate,preferably mammalian, TI gene, such as the TI sequence designated in oneof SEQ ID NOs:1-6, or a sequence complementary thereto, or naturallyoccurring mutants thereof, such that it shows more than 10 times morehybridization, preferably more than 100 times more hybridization, andeven more preferably more than 100 times more hybridization than it doesto a cellular nucleic acid (e.g., mRNA or genomic DNA) encoding aprotein other than a vertebrate, preferably mammalian, TI protein asdefined herein.

"Transcriptional regulatory sequence" is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of one of the recombinant TI genesis under the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of TI proteins.

As used herein, the term "transfection" means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. "Transformation", as used herein,refers to a piocess in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a TI polypeptide or, inthe case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the TI protein is disrupted.

As used herein, the term "transgene" means a nucleic acid sequenceencoding, e.g., one of the TI polypeptides, or an antisense transcriptthereto, which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can include one or more transcriptionalregulatory sequences and any other nucleic acid, (e.g. as intron), thatmay be necessary for optimal expression of a selected nucleic acid.

A "transgenic animal" refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the TI proteins, e.g. either agonistic or antagonisticforms. However, transgenic animals in which the recombinant TI gene issilent are also contemplated, as for example, the FLP or CRE recombinasedependent constructs described below. Moreover, "transgenic animal" alsoincludes those recombinant animals in which gene disruption of one ormore TI genes is caused by human intervention, including bothrecombination and antisense techniques.

As used herein, the term "vector" refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as"expression vectors". In general, expression vectors of utility inrecombinant DNA techniques are often in the form of "plasmids" whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, "plasmid" and "vector" are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forts of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

Nucleic Acids

As described below, one aspect of the invention pertains to isolatednucleic acids comprising nucleotide sequences encoding Tub interactor orTI polypeptides, and/or equivalents of such nucleic acids. The termequivalent is understood to include nucleotide sequences encodingfunctionally equivalent TI polypeptides or functionally equivalentpeptides having an activity of a TI protein such as described herein.Equivalent nucleotide sequences will include sequences that differ byone or more nucleotide substitution, addition or deletion, such asallelic variants; and will, therefore, include sequences that differfrom the nucleotide sequence of the TI gene shown in SEQ ID NOs:1-6 dueto the degeneracy of the genetic code.

Preferred nucleic acids are vertebrate TI nucleic acids. Particularlypreferred vertebrate TI nucleic acids are mammalian. Regardless ofspecies, particularly preferred TI nucleic acids encode polypeptidesthat are at least 80% similar to an amino acid sequence of a vertebrateTI protein. In one embodiment, the nucleic acid is a cDNA encoding apolypeptide having at least one bioactivity of the subject TIpolypeptide. Preferably, the nucleic acid includes all or a portion ofthe nucleotide sequence corresponding to the nucleic acid of SEQ IDNOs:1, 3, 5, 7, or 9.

Still other preferred nucleic acids of the present invention encode a TIpolypeptide which is comprised of at least 2, 5, 10, 25, 50, 100, 150 or200 amino acid residues. For example, preferred nucleic acid moleculesfor use as probes/primer or antisense molecules (i.e. noncoding nucleicacid molecules) can comprise at least about 6, 12, 20, 30, 50, 100, 125,150 or 200 base pairs in length, whereas coding nucleic acid moleculescan comprise about 300, 400, 500, 600, 700, 800, 900, 950, 975, 1000,1005, 1010 or 1015 base pairs.

Another aspect of the invention provides a nucleic acid which hybridizesunder stringent conditions to a nucleic acid represented by one of SEQID NOs:1-6. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0× SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0× SSC at 50° C. to a high stringency of about0.2× SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or temperature of salt concentration may be heldconstant while the other variable is changed. In a preferred embodiment,a TI nucleic acid of the present invention will bind to one of SEQ IDNOs:1-6 under moderately stringent conditions, for example at about 2.0×SSC and about 40° C. In a particularly preferred embodiment, a TInucleic acid of the present invention will bind to one of SEQ ID NOs:1-6under high stringency conditions.

Preferred nucleic acids have a sequence at least 75% homologous and morepreferably 80% and even more preferably at least 85% homologous with anamino acid sequence of a TI gene, e.g., such as a sequence shown in oneof SEQ ID NOs:1-6. Nucleic acids at least 90%, more preferably 95%, andmost preferably at least about 98-99% homologous with a nucleic sequencerepresented in one of SEQ ID NOs:1-6 are of course also within the scopeof the invention. In preferred embodiments, the nucleic acid ismammalian and in particularly preferred embodiments, includes all or aportion of the nucleotide sequence corresponding to the coding region ofone of SEQ ID NOs:1-6.

Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of SEQ ID NOs:1-6 due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., a peptide having abiological activity of a TI polypeptide) but differ in sequence from thesequence shown in the sequence listing due to degeneracy in the geneticcode. For example, a number of amino acids are designated by more thanone triplet. Codons that specify the same amino acid, or synonyms (forexample, CAU and CAC each encode histidine) may result in "silent"mutations which do not affect the amino acid sequence of a TIpolypeptide. However, it is expected that DNA sequence polymorphismsthat do lead to changes in the amino acid sequences of the subject TIpolypeptides will exist among mammalians. One skilled in the art willappreciate that these variations in one or more nucleotides (e.g., up toabout 3-5% of the nucleotides) of the nucleic acids encodingpolypeptides having an activity of a TI polypeptide may exist amongindividuals of a given species due to natural allelic variation.

As indicated by the examples set out below, TI protein-encoding nucleicacids can be obtained from mRNA present in any of a number of eukaryoticcells. It should also be possible to obtain nucleic acids encoding TIpolypeptides of the present invention from genomic DNA from both adultsand embryos. For example, a gene encoding a TI protein can be clonedfrom either a cDNA or a genomic library in accordance with protocolsdescribed herein, as well as those generally known to persons skilled inthe art. Examples of tissues and/or libraries suitable for isolation ofthe subject nucleic acids include breast, among others. A cDNA encodinga TI protein can be obtained by isolating total mRNA from a cell, e.g. avertebrate cell, a mammalian cell, or a human cell, including embryoniccells. Double stranded cDNAs can then be prepared from the total mRNA,and subsequently inserted into a suitable plasmid or bacteriophagevector using any one of a number of known techniques. The gene encodinga TI protein can also be cloned using established polymerase chainreaction techniques in accordance with the nucleotide sequenceinformation provided by the invention. The nucleic acid of the inventioncan be DNA or RNA or analogs thereof. A preferred iiucleic acid is acDNA represented by a sequence selected from the group consisting of SEQID NOs:1-6.

Vectors.

This invention also provides expression vectors containing a nucleicacid encoding a TI polypeptide, operably linked to at least onetranscriptional regulatory sequence. "Operably linked" is intended tomean that the nucleotide sequence is linked to a regulatory sequence ina manner which allows expression of the nucleotide sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe subject TI proteins. Accordingly, the term "transcriptionalregulatory sequence" includes promoters, enhancers and other expressioncontrol elements. Such regulatory sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). In one embodiment, the expression vectorincludes a recombinant gene encoding a peptide having an agonisticactivity of a subject TI polypeptide, or alternatively, encoding apeptide which is an antagonistic form of the TI protein. Such expressionvectors can be used to transfect cells and thereby produce polypeptides,including fusion proteins, encoded by nucleic acids as described herein.Moreover, the gene constructs of the present invention can also be usedas a part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of one of the subject TIproteins. Thus, another aspect of the invention features expressionvectors for in vivo or in vitro transfection and expression of a TIpolypeptide in particular cell types so as to reconstitute the functionof, or alternatively, abrogate the function of TI-induced signaling in atissue. This could be desirable, for example, when thenaturally-occurring form of the protein is misexpressed; or to deliver aform of the protein which alters differentiation of tissue. Expressionvectors may also be employed to inhibit neoplastic transformation.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a subjectTI polypeptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral targeting means of the present invention rely onendocytic pathways for the uptake of the subject TI polypeptide gene bythe targeted cell. Exemplary targeting means of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

Probes and Primers

Moreover, the nucleotide sequences determined from the cloning of TIgenes from mammalian organisms will further allow for the generation ofprobes and primers designed for use in identifying and/or cloning TIhomologues in other cell types, e.g. from other tissues, as well as TIhomologues from other mammalian organisms. For instance, the presentinvention also provides a probe/primer comprising a substantiallypurified oligonucleotide, which oligonucleotide comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast approximately 12, preferably 25, more preferably 40, 50 or 75consecutive nucleotides of sense or anti-sense sequence selected fromthe group consisting of SEQ ID NOs:1-6 or naturally occurring mutantsthereof. For instance, primers based on the nucleic acid represented inSEQ ID NOs:1, 3, 5, 7 or 9 can be used in PCR reactions to clone TIhomologues. Preferred primers for hTI-4 are set forth as SEQ ID NOs:9and 10. Preferred primers for mTI-3 are set forth as SEQ ID NOs:13 and14. Preferred primers for hTI-3 are set forth in SEQ ID NOs:17 and 18.Preferred primers for hTI-1 are set forth in SEQ ID NOs:21 and 22.Preferred primers for mTI-4 are set forth in SEQ ID NOs:25 and 26.Preferred primers for hTI-2 are set forth in SEQ ID NOs:29 and 30.

Likewise, probes based on the subject TI sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto and able to be detected, e.g. the label group isa radioisotope, a fluorescent compound, an enzyme, or an enzymeco-factor.

As discussed in more detail below, such probes can also be used as apart of a diagnostic test kit for identifying cells or tissue whichmisexpress a TI protein, such as by measuring a level of a TI-encodingnucleic acid in a sample of cells from a patient; e.g. detecting TI mRNAlevels or determining whether a genomic TI gene has been mutated ordeleted. Briefly, nucleotide probes can be generated from the subject TIgenes which facilitate histological screening of intact tissue andtissue samples for the presence (or absence) of TI-encoding transcripts.Similar to the diagnostic uses of anti-TI antibodies, the use of probesdirected to TI messages, or to genomic TI sequences, can be used forboth predictive and therapeutic evaluation of allelic mutations whichmight be manifest in, for example, a predisposition to diabetes. Used inconjunction with immunoassays as described herein, the oligonucleotideprobes can help facilitate the determination of the molecular basis fora disorder which may involve some abnormality associated with expression(or lack thereof) of a TI protein. For instance, variation inpolypeptide synthesis can be differentiated from a mutation in a codingsequence.

Antisense, Ribozyme and Triplex Techniques

Another aspect of the invention relates TO the use of the isolatednucleic acid in "antisense" therapy. As used herein, "antisense" therapyrefers to administration or in situ generation of oligonucleotidemolecules or their derivatives which specifically hybridize (e.g. bind)under cellular conditions, with the cellular mRNA and/or genomic DNAencoding one or more of the subject TI proteins so as to inhibitexpression of that protein, e.g. by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, "antisense" therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes a TI protein. Alternatively, the antisenseconstruct is an oligonucleotide probe which is generated ex vivo andwhich, when introduced into the cell causes inhibition of expression byhybridizing with the mRNA and/or genomic sequences of a TI gene. Sucholigonucleotide probes are preferably modified oligonucleotides whichare resistant to endogenous nucleases, e.g. exonucleases and/orendonucleases, and are therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by Van der Krol et al. (1988) Biotechniques6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668. With respectto antisense DNA, oligodeoxyribonucleotides derived from the translationinitiation site, e.g., between the -10 and +10 regions of the TInucleotide sequence of interest, are preferred. Particularly preferredantisense molecules are set forth in SEQ ID NOs:11, 15, 19, 23 and 27.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to TI mRNA. The antisenseoligonucleotides will bind to the TI mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. A sequence "complementary" to a portion of an RNA, as referredto herein, means a sequence having sufficient complementarity to be ableto hybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5' end of the message,e.g., the 5' untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3' untranslatedsequences of mRNAs have recently been shown to be effective atinhibiting translation of mRNAs as well. (Wagner, R. (1994) Nature372:333). Therefore, oligonucleotides complementary to either the 5' or3' untranslated, non-coding regions of a TI gene could be used in anantisense approach to inhibit translation of endogenous TI mRNA.Oligonucleotides complementary to the 5' untranslated region of the mRNAshould include the complement of the AUG start codon. Antisenseoligonucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with theinvention. Whether designed to hybridize to the 5', 3' or coding regionof TI mRNA, antisense nucleic acids should be at least six nucleotidesin length, and are preferably less that about 100 and more preferablyless than about 50, 25, 17 or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theartisense oligonucleotide to quantitate the ability of the antisenseoligonucleotide to inhibit gene expression. It is preferred that thesestudies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. It isalso preferred that these studies compare levels of the target RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. the oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO 88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalatingagents. (See, e.g, Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxyethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouricil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-idimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5'-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisensc oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.(1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2'-0-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988) Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the TI coding regionsequence can be used, those complementary to the transcribeduntranslated region are most preferred. For example, an antisenseoligonucleotide as set forth in SEQ ID NOs:11, 15, 19, 23 and 27 can beutilized in accordance with the invention.

The antisense molecules should be delivered to cells which express TI invivo. A number of methods have been developed for delivering antisenseDNA or RNA to cells; e.g., antisense molecules can be injected directlyinto the tissue site, or modified antisense molecules, designed totarget the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systematically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation on endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous TI transcripts and therebyprevent translation of the TI mRNA. For example, a vector can beintroduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bemoist and Chambon (1981) Nature 290:304-310),the promoter contained in the 3' long terminal repeat of Rous sarcomavirus (Yamamoto et al. (1980) Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al. (1982) Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site; e.g.,the choroid plexus or hypothalamus. Alternatively, viral vectors can beused which selectively infect the desired tissue; (e.g., for brain,herpesvirus vectors may be used), in which case administration may beaccomplished by another route (e.g., systematically).

Ribozyme molecules designed to catalytically cleave TI mRNA transcriptscan also be used to prevent translation of TI mRNA and expression of TI(See, e.g., PCT Publication No. WO 90/11364, published Oct. 4, 1990;Sarver et al. (1990) Science 247:1222-1225 and U.S. Pat. No. 5,093,246).While ribozymes that cleave mRNA at site specific recognition sequencescan be used to destroy TI mRNAs, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5'-UG-3'. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach (1988) Nature 334:585-591. There arehundreds of potential hammerhead ribozyme cleavage sites within thenucleotide sequence of human TI cDNA (FIG. 1). Preferably the ribozymeis engineered so that the cleavage recognition site is located near the5' end of the TI mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter "Cech-type ribozymes") such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al. (1984) Science 224:574-578; Zaug andCech (1986) Science 231:470-475; Zaug, et al. (1986) Nature 324:429-433;published PCT Publication No. WO 88/04300 by University Patents Inc.;Been and Cech, (1986) Cell 47:207-216). The Cech-type ribozymes have aneight base pair active site which hybridizes to a target RNA sequencewhereafter cleavage of the target RNA takes place. The inventionencompasses those Cech-type ribozymes which target eight base-pairactive site sequences that are present in a TI gene. Particularlypreferred ribozymes are set forth in SEQ ID NOs:8, 12, 16, 20, 24 and28.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) andshould be delivered to cells which express the TI gene in vivo. Apreferred method of delivery involves using a DNA construct "encoding"the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous TI messages and inhibittranslation. Because ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Endogenous TI gene expression can also be reduced by inactivating or"knocking out" the TI gene or its promoter using targeted homologousrecombination. (see, e.g, Smithies et al. (1985) Nature 317:230-234;Thomas and Capecchi (1987) Cell 51:503-512; Thompson et al. (1989) Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional TI (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenous TIgene (either the coding regions or regulatory regions of the TI gene)can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express TI in vivo. Insertionof the DNA construct, via targeted homologous recombination, results ininactivation of the TI gene. Such approaches are particularly suited inthe agricultural field where modifications to ES (embryonic stem) cellscan be used to generate animal offspring with an inactive TI (e g., seeThomas and Capecchi, 1987, and Thompson, 1989, supra). However thisapproach can be adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors, e.g., herpes virus vectors fordelivery to brain tissue; e.g., the hypothalamus and/or choroid plexus.

Alternatively, endogenous TI gene expression can be reduced by targetingdeoxyribonucleotide sequences complementary to the regulatory region ofthe TI gene (i.e., the TI promoter and/or enhancers) to form triplehelical structures that prevent transcription of the TI gene in targetcells in the body. (See generally, Helene, C. (1991) Anticancer DrugDes. 6(6):569-84; Helene, C., et al. (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15).

Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of one of the TI proteins,can be used in the manipulation of issue, e.g. lipid metabolism, both invivo and for ex vivo tissue cultures.

Furthermore, like the antisense techniques (e.g. microinjection ofantisense molecules, or transfection with plasmids whose transcripts areantisense with regard to a TI mRNA or gene sequence) antagonizing thenormal biological activity of one of the TI proteins can be used toinvestigate role of TI in lipid metabolism. Such techniques can beutilized in cell culture, but can also be used in the creation oftransgenic animals, as detailed below.

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called "switchback"nucleic acid molecule. Switchback molecules are synthesized in analternating 5'-3', 3'-5' manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Antisense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramide chemical synthesis. Alternatively, RNA molecules maybe generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polyinerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5' and/or 3' ends of the molecule or the useof phosphorothioate or 2' O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

Polypeptides of the Invention

The present invention also makes available isolated TI polypeptideswhich are isolated from, or otherwise substantially free of othercellular proteins, especially other signal transduction factors and/ortranscription factors which may normally be associated with the TIpolypeptide. The term "substantially free of other cellular proteins"(also referred to herein as "contaminating proteins") or "substantiallypure or purified preparations" are defined as encompassing preparationsof TI polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein. By "purified", it is meant, whenreferring to a peptide or DNA or RNA sequence, that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules, such as other proteins. The term "purified" as usedherein preferably means at least 80% by dry weight, more preferably inthe range of 95-99% by weight, and most preferably at least 99.8% byweight, of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term "pure" asused herein preferably has the same numerical limits as "purified"immediately above. "Isolated" and "purified" do not encompass eithernatural materials in their native state or natural materials that havebeen separated into components (e.g., in an acrylamide gel) but notobtained either as pure (e.g. lacking contaminating proteins, orchromatography reagents such as denaturing agents and polymers, e.g.acrylamide or agarose) substance, or solutions. In preferredembodiments, purified TI preparations will lack any contaminatingproteins from the same animal from which TI is normally produced, as canbe accomplished by recombinant expression of, for example, a human TIprotein in a non-human cell.

Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 25, 50, 75, 100, 125, 150 amino acids in length are withinthe scope of the present invention.

For example, isolated TI polypeptides can be encoded by all or a portionof a nucleic acid sequence shown in any of SEQ ID NOs:1-6. Isolatedpeptidyl portions of TI proteins can be obtained by screening peptidesrecombinantly produced from the corresponding fragment of the nucleicacid encoding such peptides. In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. For example, a TIpolypeptide of the present invention may be arbitrarily divided intofragments of desired length with no overlap of the fragments, orpreferably divided into overlapping fragments of a desired length. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments which can function as eitheragonists or antagonists of a wild-type (e.g., "authentic") TI protein.

Another aspect of the present invention concerns recombinant forms ofthe TI proteins. Recombinant polypeptides preferred by the presentinvention, in addition to native TI proteins, are encoded by a nucleicacid, which is at least 85% homologous and more preferably 90%homologous and most preferably 95% homologous with an amino acidsequence represented by SEQ ID NOs:1-6. Polypeptides which are encodedby a nucleic acid that is at least about 98-99% homologous with thesequence of SEQ ID NOs: 1-6 are also within the scope of the invention.In a preferred embodiment, a TI protein of the present invention is amammalian TI protein. In a particularly preferred embodiment a TIprotein is encoded by one of the nucleic acids set forth as SEQ IDNOs:1-6. In particularly preferred embodiment, a TI protein has a TIbioactivity. It will be understood that certain post-translationalmodifications, e.g., phosphorylation and the like, can increase theapparent molecular weight of the TI protein relative to the unmodifiedpolypeptide chain.

The present invention further pertains to recombinant forms of one ofthe subject TI polypeptides. Such recombinant TI polypeptides preferablyare capable of functioning in one of either role of an agonist orantagonist of at least one biological activity of a wildtype("authentic") TI protein of the appended sequence listing. The term"evolutionarily related to", with respect to amino acid sequences of TIproteins, refers to both polypeptides having amino acid sequences whichhave arisen naturally, and also to mutational variants of human TIpolypeptides which are derived, for example, by combinatorialmutagenesis.

In general, polypeptides referred to herein as having an activity (e.g.,are "bioactive") of a TI protein are defined as polypeptides whichinclude an amino acid sequence encoded by all or a portion of thenucleic acid sequences shown in one of SEQ ID NOs:1-6 and which mimic orantagonize all or a portion of the biological/biochemical activities ofa naturally occurring TI protein. In preferred embodiments a TI proteinof the present invention specifically interacts with a the carboxyterminus (i.e. last 44 amino acids) of the human tub polypeptide.Examples of such biological activity include the ability to modulateweight control/or diabetes. Other biological activities of the subjectTI proteins are described herein or will be reasonably apparent to thoseskilled in the art. According to the present invention, a polypeptidehas biological activity if it is a specific agonist or antagonist of anaturally-occurring form of a TI protein.

The present invention further pertains to methods of producing thesubject TI polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. The cells may beharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The recombinant TI polypeptide can be isolatedfrom cell culture medium, host cells, or both using techniques known inthe art for purifying proteins including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for such peptide.In a preferred embodiment, the recombinant TI polypeptide is a fusionprotein containing a domain which facilitates its purification, such asGST fusion protein.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologues of one ofthe subject TI polypeptides which function in a limited capacity as oneof either a TI agonist (mimetic) or a TI antagonist, in order to promoteor inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homologue of limitedfunction, and with fewer side effects relative to treatment withagonists or antagonists which are directed to all of the biologicalactivities of naturally occurring forms of TI proteins.

Homologues of each of the subject TI proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologues which retainsubstantially the same, or merely a subset, of the biological activityof the TI polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to a downstream or upstream member ofthe TI cascade which includes the TI protein. In addition, agonisticforms of the protein may be generated which are constitutively active.Thus, the TI protein and homologues thereof provided by the subjectinvention may be either positive or negative regulators of weightcontrol and/or diabetes.

The recombinant TI polypeptides of the present invention also includehomologues of the wild-type TI proteins, such as versions of thoseprotein which are resistant to proteolytic cleavage, as for example, dueto mutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

TI polypeptides may also be chemically modified to create TI derivativesby forming covalent or aggregate conjugates with other chemicalmoieties, such as glycosyl groups, lipids, phosphate, acetyl groups andthe like. Covalent derivatives of TI proteins can be prepared by linkingthe chemical moieties to functional groups on amino acid sidechains ofthe protein or at the N-terminus or at the C-terminus of thepolypeptide.

Modification of the structure of the subject TI polypeptides can be forsuch purposes as enhancing therapeutic or prophylactic efficacy,stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo), or post-translational modifications (e.g., toalter phosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring lormof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the TI polypeptides described inmore detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect ihat an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids are can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer,W. H. Freeman and Co., 1981). Whether a change in the amino acidsequence of a peptide results in a functional TI homologue (e.g.functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a respoise in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject TI proteins as well as truncationmutants, and is especially useful for identifying potential variantsequences (e.g. homologues) that are functional in modulating signaltransduction from a lipid receptor. The purpose of screening suchcombinatorial libraries is to generate, for example, novel TI homologueswhich can act as either agonists or antagonist, or alternatively,possess novel activities all together. To illustrate, TI homologues canbe engineered by the present method to provide selective, constitutiveactivation of a tub signaling pathway. Thus, combinatorially-derivedhomologues can be generated to have an increased potency relative to anaturally occurring form of the protein.

Likewise, TI homologues can be generated by the present combinatorialapproach to selectively inhibit (antagonize) induction by a lipid. Forinstance, mutagenesis can provide TI homologues which are able to bindother signal pathway proteins (or DNA) yet prevent propagation of thesignal, e.g. the homologues can be dominant negative mutants. Moreover,manipulation of certain domains of TI by the present method can providedomains more suitable for use in fusion proteins.

In one embodiment, the variegated library of TI variants is generated bycombinatorial mutagenesis at the nucleic acid level, and is encoded by avariegated gene library. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential TI sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g. for phage display) containing the set of TI sequencestherein.

There are many ways by which such libraries of potential TI homologuescan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential TI sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) Proc. Natl. Acad. Sci. USA 89:2429-2433; Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) Proc. Natl. Acad.Sci. USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5.198,346,and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for aTI clone in order to generate a variegated population of TI fragmentsfor screening and subsequent selection of bioactive fragments. A varietyof techniques are known in the art for enerating such libraries,including chemical synthesis. In one embodiment, a library of codingsequence fragments can be generated by (i) treating a double strandedPCR fragment of a TI coding sequence with a nuclease under conditionswherein nicking occurs only about once per molecule; (ii) denaturing thedouble stranded DNA; (iii) renaturing the DNA to form double strandedDNA which car include sense/antisense pairs from different nickedproducts; (iv) removing single stranded portions from reformed duplexesby treatment with S1 nuclease; and (v) ligating the resulting fragmentlibrary into an expression vector. By this exemplary method, anexpression library can be derived which codes for N-terninal, C-terminaland internal fragments of various sizes.

A wide range of techniques arc known in the art for screening geneproducts of combinatorial libraries made by point mutatiors ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of TI homologues. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate TI sequences created bycombinatorial mutagenesis techniques.

In one embodiment, cell based assays can be exploited to analyze thevariegated TI library. For instance, the library of expression vectorscan be transfected into a cell line ordinarily responsive to insulin.The transfected cells are then contacted with the insulin and the effectof the TI mutant on signaling by a Y5 receptor can be detected. PlasmidDNA can then be recovered from the cells which score for inhibition, oralternatively, potentiation of lipid receptor induction, and theindividual clones further characterized.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays. To overcome thisproblem, a new technique has been developed recently, recrusive ensemblemutagenesis (REM), which allows one to avoid the very high proportion ofnon-functional proteins in a random library and simply enhances thefrequency of functional proteins, thus decreasing the complexityrequired to achieve a useful sampling of sequence space. REM is analgorithm which enhances the frequency of functional mutants in alibrary when an appropriate selection or screening method is employed(Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;Yourvan et al. (1992) Parallel Problem Solving from Nature, 2., InMaenner and Manderick, eds., Elsevier Publishing Co., Amsterdam, pp.401-410; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

The invention also provides for reduction of the TI proteins to generatemimetics, e.g. peptide or non-peptide agents, which are able to disruptbinding of a TI polypeptide of the present invention with eitherupstream or downstream components of a lipid uptake signaling cascade,such as binding proteins or interactors. Thus, such mutagenic techniquesas described above are also useful to map the determinants of the TIproteins which participate in protein-protein interactions involved in,for example, binding of the subject TI polypeptide to proteins which mayfunction upstream (including both activators and repressors of itsactivity) or to proteins or nucleic acids which may function downstreamof the TI polypeptide, whether they are positively or negativelyregulated by it, for example. To illustrate, the critical residues of asubject TI polypeptide which are involved in molecular recognition of,for example, tub or other components upstream or downstream of a TI canbe determined and used to generate TI-derived peptidomimetics whichcompetitively inhibit binding of the authentic TI protein with thatmoiety. By employing, for example, scanning mutagenesis to map the aminoacid residues of each of the subject TI proteins which are involved inbinding other extracellular proteins, peptidomimetic compounds can begenerated which mimic those residues of the TI protein which facilitatethe interaction. Such mimetics may then be used to interfere with thenormal function of a TI protein. For instance, non-hydrolyzable peptideanalogs of such residues can be generated using benzodiazepine (e.g.,see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopepi ides (Ewenson et al. (1986) J. Med. Chem. 29:295; and Ewensonet al. in Peptides: Structure and Function (Proceedings of the 9thAmerican Peptide Symposium) Pierce Chemical Co. Rockland, Ill, 1985),b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; andSato et al. (1986) J. Chem. Soc. Perkin. Trans. 1:1231), andb-aminoalcohols (Gordon et al. (1985) Biochem. Biophys. Res. Commun.126:419; and Dann et al. (1986) Biochem. Biophys. Res. Commun. 134:71).

Cells Expressing Recombinant TI Polypeptides.

This invention also pertains to host cells transfected to express arecombinant form of the subject TI polypeptides. The host cell may beany prokaryotic or eukaryotic cell. Thus, a nucleotide sequence derivedfrom the cloning of mammalian TI proteins, encoding all or a selectedportion of the full-length proteir, can be used to produce a recombinantform of a TI polypeptide via microbial or eukaryotic cellular processes.Ligating the polynucleotide sequence into a gene construct, such as anexpression vector, and transforming or transfecting into hosts, eithereukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterialcells), are standard procedures used in producing other well-knownproteins, e.g. MAP kinase, p53, WT1, PTP phosphatases, SRC, and thelike. Similar procedures, or modifications thereof, can be employed toprepare recombinant TI polypeptides by microbial means or tissue-culturetechnology in accord with the subject invention.

The recombinant TI genes can be produced by ligating a nucleic acidencoding a TI protein, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells, or both.Expression vectors for production of recombinant forms of the subject TIpolypeptides include plasmids and other vectors. For instance, suitablevectors for the expression of a TI polypeptide include plasmids of thetypes: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derivedplasmids, pBTac-derived plasmids and pUC-derived plasmids for expressionin prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach el al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a TI polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the TI genes represented in SEQ ID NOs:1,3, 5, 7, or 9.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, PKO-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and cukaryotic cells.Alternatively. derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant TIpolypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL94 1), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

When it is desirable to express only a portion of a TI protein, such asa form lacking a portion of the N-terminus, i.e. a truncation mutantwhich lacks the signal peptide, it may be necessary to add a start codon(ATG) to the oligonucleotide fragment containing the desired sequence tobe expressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) Proc. Natl. Acad. Sci.84:2718-1722). Therefore, removal of an N-terminal methionine, ifdesired, can be achieved either in vivo by expressing TI-derivedpolypeptides in a host which produces MAP (e.g., E. coli or CM89 or S.cerevisiae), or in vitro by use of purified MAP (e.g., procedure ofMiller et al., supra).

In other embodiments transgenic animal, described in more detail belowcould be used to produce recombinant proteins.

Fusion Proteins and Immunogens.

In another embodiment, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of a TI protein. For example, the VP6 capsid protein ofrotavirus can be used as an immunologic carrier protein for portions ofthe TI polypeptide, either in the monomeric form or in the form of aviral particle. The nucleic acid sequences corresponding to the portionof a subject TI protein to which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising TI epitopesas part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the hepatitis b surface antigenfusion proteins that recombinant hepatitis b virions can be utilized inthis role as well. Similarly, chimeric constructs coding for fusionproteins containing a portion of a TI protein and the poliovirus capsidprotein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Plublication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofa TI polypeptide is obtained directly from organo-chemical synthesis ofthe peptide onto an oligomeric branching lysine core (see, for example,Posnett et al. (1988) J. Biol. Chem. 263:1719 and Nardelli et al. (1992)J. Immunol. 148:914). Antigenic determinants of TI proteins can also beexpressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, and accordingly, can be used in the expressionof the TI polypeptides of the present invention. For example, TIpolypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins. Such GST-fusion proteins can enable easy purification of theTI polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (John Wiley & Sons, NY 1991)).

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni2+ metal resin. The purification leadersequence can then be subsequently removed by treatment witn enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Antibodies

Another aspect of the invention pertains to an antibody specificallyreactive with a mammalian TI protein. For example, by using immunogensderived from a TI protein, e.g. based on the cDNA sequences,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols (See, for example, Antibodies: A Laboratory Manualed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, suchas a mouse, a hamster or rabbit can be immunized with an immunogenicform of the peptide (e.g., a ammalian TI polypeptide or an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein as described above). Techniques for conferring immunogenicity ona protein or peptide include conjugation to carriers or other techniqueswell known in the art. An immunogenic portion of a TI protein can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies. In a preferred embodiment,the subject antibodies are immunospecific for antigenic determinants ofa TI protein of a mammal, e.g. antigenic determinants of a proteinencoded by SEQ ID NOs:1-6 or closely related homologues (e.g. at least900% homologous, and more preferably at least 94% homologous).

Following immunization of an animal with an antigenic preparation of aTI polypeptide, anti-TI antisera can be obtained and, if desired,polyclonal anti-TI antibodies isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature 256: 495-497), the human B cellhybridoma technique (Kozbar et al. (1983) Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian TIpolypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells. In one embodimentanti-human TI antibodies specifically react with any of the proteinsencoded by the DNA of ATCC deposit Nos. 98125-98128.

The term "antibody" as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian TI polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to inchlde bispecific, single-chain and chimeric moleculeshaving affinity for a TI protein conferred by at least one CDR region ofthe antibody. In preferred embodiments, the antibody further comprises alabel attached thereto and able to be detected, (e.g. the label can be aradioisotope, fluorescent compound, enzyme or enzyme co-factor).

Antibodies which specifically bind TI epitopes can also be used inimmunohistochemical staining of tissue samples in order to evaluate theabundance and pattern of expression of each of the subject TIpolypeptides. Anti-TI antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate TIprotein levels in tissue as part of a clinical testing procedure. Forinstance, such measurements can be useful in predictive valuations ofthe onset or progression of proliferative disorders. Likewise, theability to monitor TI protein levels in an individual can allowdetermination of the efficacy of a given treatment regimen for anindividual afflicted with such a disorder. The level of TI polypeptidesmay be measured from cells in bodily fluid, such as in samples ofcerebral spinal fluid, such as produced by biopsy. Diagnostic assaysusing anti-Ti antibodies can include, for example, immunoassays designedto aid in early diagnosis of a degenerative disorder. Diagnostic assaysusing anti-TI polypeptide antibodies can also include immunoassaysdesigned to aid in early diagnosis and phenotyping neoplasic orhyperplastic disorders.

Another application of anti-TI antibodies of the present invention is inthe immunological screening of cDNA libraries constructed in expressionvectors such as λ gt11, λgt18-23, λZAP, and λORF8. Messenger librariesof this type, having coding sequences inserted in the correct readingframe and orientation, can produce fusion proteins. For instance, λgt11will produce fusion proteins whose amino termini consist ofβ-galactosidase amino acid sequences and whose carboxy termini consistof a foreign polypeptide. Antigenic epitopes of a TI protein, e.g. otherorthologues of a particular TI protein or other paralogues from the samespecies, can then be detected with antibodies, as, for example, reactingnitrocellulose filters lifted from infected plates with anti-TIantibodies. Positive phage detected by this assay can then be isolatedfrom the infected plate. Thus, the presence of TI homologues can bedetected and cloned from other animals, as can alternate isoforms(including splicing variants) from humans.

Methods of Treating Disease

There are a wide variety of disorders for which TI molecules of thepresent invention can be used in treatment. As discussed herein TImolecule can increase the transcription or activity of TI molecules in acell. A TI molecule therapeutic can be, as appropriate, any of thepreparations described above, including isolated polypeptides, genetherapy constructs, antisense molecules, peptidomimetics or agentsidentified in the drug assays provided herein.

In preferred embodiments the subject TI molecules are modulated tocontrol weight in a subject. Hypothalamic neuropeptide Y (NPY) is amember of the pancreatic polypeptide family and is a potent feedingsignal. NPY levels in the paraventricular nucleus (PVN) of the brainhave been shown to increase with food deprivation and return to normalafter insulin injections (Sahu et al. (1995) Endocrinology 136:5718). Inone embodiment the subject TI molecules are modulated to control weightin a subject by modulation of a biochemical pathway involving NPY. NPYis thought to signal via the Y5 receptor (Gerald et al. (1996) Nature382:168). The distribution of Y5 mRNA shows that the Y5 receptor is alsoinvolved in regulating the emotional aspect of appetitive behaviors. Inanother embodiment the subject TI molecules are modulated to controlweight by modulation of a biochemical pathway involving the Y5 receptor.

Insulin regulates food intake by altering NPY expression in thehypothalamus of the brain (Schwartz et al. (1992) Endocr Rev. 13:387).Insulin deficiency, which can be caused, for example, by diabetes, isthought to lead to increased NPY expression in the hypothalamus and tothe hyperphagia characteristic of uncontrolled type I diabetes (Sipolset al. (1995) Diabetes 44:147). In one embodiment the subject TImolecules are modulated to control weight in a subject by modulation ofa biochemical pathway involving insulin. In another embodiment, obesityis controlled by modulation of a biochemical pathway involvinginsulin-like growth factor II (IGF-II).

In other embodiments, the subject TI molecules are modulated to affect abioactivity of tub in order to effect a treatment for weight control. Ina preferred embodiment the subject TI molecules are modulated to controlobesity, diabetes, or cachexia.

In still other embodiments, the subject TI molecules are modulated tocontrol apoptosis in a cell. Apoptosis, or programmed cell death, ischaracterized by distinct morphological changes and can be triggered bya variety of mechanisms. Certain apoptosis-inducing agents stimulatesphingomyelinases, which act on sphingolipids resulting in thegeneration of phosphocholine and ceramide, a key regulator of cell cyclecontrol and apoptosis (Pushkareva et al. (1995) Immunology Today16:295). Ceramide is thought to act as a second messenger since asoluble analog of ceramide mimics the affects of agents that induceceramide production (Law and Rossie (1995) J. Biol Chem. 270:12808).Ceramide is thought to control apoptosis via its interaction with theprotein phosphatase 2A (PP2A) family of serine/threonine proteinphosphatases (Hannun (1994) J. Biol. Chem. 269:3125). The catalyticsubunit of PP-2A has been shown to be activated by Ceramide (Law andRossie, supra).

In a preferred embodiment the subject TI molecules are modulated tocontrol apoptosis in a cell of the PVN of the brain. In one embodimentmodulation of the molecules to control apoptosis in the PVN of the brainleads to one or more of weight control and diabetes in a subject.

In one embodiment apoptosis is modulated by modulating the activity ofTI-1 in a cell. In yet another embodiment apoptosis is modulated bymodulating the activity of TI-2 in a cell. In still another embodimentapoptosis is modulated by modulating the activity of TI-3 in a cell. Inanother embodiment apoptosis is modulated by modulating TI-4 activity ina cell. In addition, therapy may involve modulation of any combinationof the disclosed TI molecules.

The present invention will also be useful in treating neurodegenerativediseases which are characterized by apoptosis, including Alzheimer'sdisease, Parkinson's disease, Huntington's chorea, amylotrophic lateralsclerosis and the like, as well as spinocerebellar degenerations.

In another embodiment the present invention can be used to modulate apathway involving integrin-mediated signaling.

In another embodiment the subject TI molecules are modulated to controlcell cycle progression. Entry of cells into mitosis characteristicallyinvolves coordinated and simultaneous events, which include, forexample, cytoskeletal rearrangements, disassembly of the nuclearenvelope and of the nucleoli, and condensation of chromatin intochromosomes. Cell-cycle events are thought to be regulated by a seriesof interdependent biochemical steps, with the initiation of late eventsrequiring the successful completion of those proceeding them. Ineukaryotic cells mitosis does not normally take place until the G1, Sand G2 phases of the cell-cycle are completed. For instance, at leasttwo stages in the cell cycle are regulated in response to DNA damage,the G1/S and the G2/M transitions. These transitions serve ascheckpoints to which cells delay cell-cycle progress to allow repair ofdamage before entering either S phase, when damage would be perpetuated,or M phase, when breaks would result in loss of genomic material. Boththe G1/S and G2/M checkpoints are known to be under genetic control asthere are mutants that abolish arrest or delay which ordinarily occur inwild-type cells in response to DNA damage.

Tumor suppressors have also been linked to cell cycle control. Forexample, both p53 (Green (1989) Cell 56:1-3; Mowat et al (1985) Nature314:633-636) and the retinoblastoma gene produce (Rb) have been linkedto cell cycle control. The first firm evidence for a specificbiochemical link between p53 and the cell cycle comes a finding that p53apparently regulates expression of a second protein, p21, which inhibitscyclin-dependent kinases (cdks) needed to drive cells through the cellcycle, e.g. from G1 into S phase (Xiong et al. (1993) Nature366:701-704). C6 ceramide has been shown to cause dephosphorylation ofRb and Rb deficient cells are more resistant to ceramide-induced growthsuppression (Pushkareva et al. supra).

In one embodiment cell cycle progression is modulated by modulating theactivity of TI-1 in a cell. In yet another embodiment cell cycleprogression is modulated by modulating the activity of TI-2 in a cell.In still another embodiment cell cycle progression is modulated bymodulating the activity of TI-3 in a cell. In another embodiment cellcycle progression is modulated by modulating TI-4 in a cell. Inaddition, therapy may involve modulation of any combination of thedisclosed TI molecules.

Since, in some cases, genes may be upregulated in a disease state and inother cases may be suppressed, it will be desirable to activate and/orpotentiate or suppress and/or downmodulate TI bioactivity depending onthe condition to be treated using the techniques compounds and methodsdescribed herein.

Among the approaches which may be used to ameliorate disease symptomsinvolving the misexpression of a TI gene are, for example, antisense,ribozyme, and triple helix molecules described above. Compounds thatcompete with an TI protein for binding to upstream or downstreamelements in a lipid uptake signaling cascade will antagonize a TIprotein, thereby inducing a therapeutic effect. Examples of suitablecompounds include the antagonists or homologues described in detailabove. In other instances, the increased expression or bioactivity of aTI protein may be desirable and may be accomplished by, for example theuse of the TI agonists or mimetics or by gene replacement therapy, asdescribed herein.

Compounds identified as increasing or decreasing TI gene expression orprotein activity can be administered to a subject at therapeuticallyeffective dose to treat the diseases described herein. A therapeuticallyeffective dose refers to that amount of the compound sufficient toeffect a change in a TI-associated disorder, such as abnormal weightcontrol and/or diabetes.

Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining The LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the oligomers of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the oligomers of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the oligomers maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a nietered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g. in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art.

In clinical settings, the gene delivery systems for the therapeutic TIgene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g. by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). A TI gene, such as anyone of the sequences represented in the group consisting of SEQ IDNOs:1-6 or a sequence homologous thereto can be delivered in a genetherapy construct by electroporation using techniques described, forexample, by Dev et al. ((1994) Cancer Treat. Rev. 20:105-115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Diagnostic and Prognostic Assays

In the diagnostic and prognostic assays described herein, in addition tothe TI nucleic acid molecules and polypeptides described above, thepresent invention provides for the use of a nucleic acid comprising atleast a portion of a TI nucleic acid molecule, for example, at least aportion of a nucleic acid sequence shown in SEQ ID NOs:1-6 orpolypeptides encoded by at least a portion of the nucleic acid sequenceshown in SEQ ID NOs:1-6.

The present method provides a method for determining if a subject is atrisk for a disorder characterized by apoptosis or aberrant cellproliferation. In preferred embodiments, the methods can becharacterized as comprising detecting, in a sample of cells from thesubject, the presence or absence of a genetic lesion characterized by atleast one of (i) an alteration affecting the integrity of a geneencoding a TI-protein, or (ii) the mis-expression of the TI gene. Toillustrate, such genetic lesions can be detected by ascertaining theexistence of at least one of (i) a deletion of one or more nucleotidesfrom a TI gene, (ii) an addition of one or more nuclectides to a TIgene, (iii) a substitution of one or more nucleotides of a TI gene, (iv)a gross chromosomal rearrangement of a TI gene, (v) a gross alterationin the level of a messenger RNA transcript of a TI gene, (vii) aberrantmodification of a TI gene, such as of the methylation pattern of thegenomic DNA, (vii) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a TI gene, (viii) a non-wild type level of aTI-protein, (ix) allelic loss of a TI gene, and (x) inappropriatepost-translational modification of a TI-protein. As set out below, thepresent invention provides a large number of assay techniques fordetecting lesions in a TI gene, and importantly, provides the ability todiscern between different molecular causes underlying TI-dependentaberrant cell growth, proliferation and/or differentiation.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a (purified) oligonucleotide probe including a region ofnucleotide sequence which is capable of hybridizing to a sense orantisense sequence of a TI gene, such as represented by any of SEQ IDNOs:1-6, or naturally occurring mutants thereof, or 5' or 3' flankingsequences or intronic sequences naturally associated with the subject TIgenes or naturally occurring mutants thereof. The nucleic acid of a cellis rendered accessible for hybridization, the probe is exposed tonucleic acid of the sample, and the hybridization of the probe to thesample nucleic acid is detected. Such techniques can be used to detectlesions at either the genomic or mRNA level, including deletions,substitutions, etc., as well as to determine mRNA transcript levels.

As set out above, one aspect of the present invention relates todiagnostic assays for determining, in the context of cells isolated froma patient, if mutations have arisen in one or more TI of the samplecells. The present method provides a method for determining if a subjectis at risk for a disorder characterized by aberrant cell proliferationand/or differentiation. In preferred embodiments, the method can begenerally characterized as comprising detecting, in a sample of cellsfrom the subject, the presence or absence of a genetic lesioncharacterized by an alteration affecting the integrity of a geneencoding a TI. To illustrate, such genetic lesions can be detected byascertaining the existence of at least one of (i) a deletion of one ormore nucleotides from a TI-gene, (ii) an addition of one or morenucleotides to a TI-gene, (iii) a substitution of one or morenucleotides of a TI-gene, and (iv) the presence of a non-wild typesplicing pattern of a messenger RNA transcript of a TI-gene. As set outbelow, the present invention provides a large number of assay techniquesfor detecting lesions in TI genes.

In certain embodiments, detection of the lesion comprises utilizing theprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the TI-gene (see Abravaya et al. (1995) NucAcid Res 23:675-682). In a merely illustrative embodiment, the methodincludes the steps of (i) collecting a sample of cells from a patient,(ii) isolating nucleic acid (c .g., genomic, mRNA or both) from thecells of the sample, (iii) contacting the nucleic acid sample with oneor more primers which specifically hybridize to a TI gene underconditions such that hybridization and amplification of the TI-gene (ifpresent) occurs, and (iv) detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Another embodiment of the invention provides for a nucleic acidcomposition comprising a (purified) oligonucleotide probe including aregion of nucleotide sequence which is capable of hybridizing to a senseor antisense sequence of a TI-gene, or naturally occurring mutantsthereof, or 5' or 3' flanking sequences or intronic sequences naturallyassociated with the subject TI-genes or naturally occurring mutantsthereof. The nucleic acid of a cell is rendered accessible forhybridization, the probe is exposed to nucleic acid of the sample, andthe hybridization of the probe to the sample nucleic acid is detected.Such techniques can be used to detect lesions at either the genomic ormRNA level, including deletions, substitutions, etc., as well as todetermine mRNA transcript levels. Such oligonucleotide probes can beused for both predictive and therapeutic evaluation of allelic mutationswhich might be manifest in, for example, apoptosis or aberrant cellgrowth.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a TI gene.

Antibodies directed against wild type or mutant TI proteins, which arediscussed, above, may also be used in disease diagnostics andprognostics. Such diagnostic methods, may be used to detectabnormalities in the level of TI protein expression, or abnormalities inthe structure and/or tissue, cellular, or subcellular location of TIprotein. Structural differences may include, for example, differences inthe size, electronegativity, or antigenicity of the mutant TI proteinrelative to the normal TI protein. Protein from the tissue or cell typeto be analyzed may easily be detected or isolated using techniques whichare well known to one of skill in the art, including but not limited towestern blot analysis. For a detailed explanation of methods forcarrying out western blot analysis, see Sambrook et al, 1989, supra, atChapter 18. The protein detection and isolation methods employed hereinmay also be such as those described in Harlow and Lane, for example,(Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of TI proteins. In situdetection may be accomplished by removing a histological specimen from apatient, and applying thereto a labeled antibody of the presentinvention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the TI protein, but also its distribution in theexamined tissue. Using the present invention, one of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an anti-TI protein specific antibody is vialinkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller,"The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md.; Voller, et al., J. Clin. Pathol. 31:507-520 (1978);Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) EnzymeImmunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.)Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵² Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. Likewise, a bioluminescent compound may be used to labelthe antibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

Moreover, it will be understood that any of the above methods fordetecting alterations in a TI gene or gene product can be used tomonitor the course of treatment or therapy.

Drug Screening Assays

In drug screening assays described herein, in addition to the TI nucleicacid molecules and polypeptides described above, the present inventionalso provides for the use of nucleic acid molecules comprising at leasta portion of a TI nucleic acid molecule, for example, at least a portionof a sequence shown in SEQ ID NOs:1-6 or polypeptides encoded by atleast a portion of the nucleic acid sequence shown in any of SEQ IDNOs:1-6.

Furthermore, by making available purified and recombinant TIpolypeptides, the present invention facilitates the development ofassays which can be used to screen for drugs, including homologues,which are either agonists or antagonists of the normal cellular functionof the subject polypeptides. In one embodiment, the assay evaluates theability of a compound to modulate binding between a TI polypeptide and amolecule, be it protein or DNA, that interacts either upstream ordownstream of the TI polypeptide in a lipid transfer pathway. A varietyof assay form,ts will suffice and, in light of the present inventions,will be comprehended by a skilled artisan.

Cell-Free Assays

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as"primary" screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements. Accordingly, in an exemplary screening assay of thepresent invention, the compound of interest is contacted with proteinswhich may function upstream (including both activators and repressors ofits activity) or to proteins or nucleic acids which may functiondownstream of the TI polypeptide, whether they are positively ornegatively regulated by it. To the mixture of the compound and theupstream or downstream element is then added a composition containing aTI polypeptide. Detection and quantification of complexes of TI withit's upstream or downstream elements provide a means for determining acompound's efficacy at inhibiting (or potentiating) complex formationbetween TI and the TI-binding elements. The efficacy of the compound canbe assessed by generating dose response curves from data obtained usingvarious concentrations of the test compound. Moreover, a control assaycan also be performed to provide a baseline for comparison. In thecontrol assay, isolated and purified TI polypeptide is added to acomposition containing the TI-binding element, and the formation of acomplex is quantitated in the absence of the test compound.

Complex formation between the TI polypeptide and a binding element(e.g., Tub) may be detected by a variety of techniques. Modulation ofthe formation of complexes can be quantitated using, for example,detectably labeled proteins such as radiolabeled, fluorescently labeled,or enzymatically labeled TI polypeptides, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either TI or its bindingprotein to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of TI to an upstream or downstream element, in thepresence and absence of a candidate agent, can be accomplished in anyvessel suitable for containing the reactants. Examples includemicrotitre plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows the protein to be bound to a matrix. For example,glutathione-S-transferase/TI (GST/TI) fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates, e.g. an ³⁵ S-labeled, and the test compound, and themixture incubated under conditions conducive to complex formation, e.g.at physiological conditions for salt and pH, though slightly morestringent conditions may be desired. Following incubation, the beads arewashed to remove any unbound label, and the matrix immobilized andradiolabel determined directly (e.g. beads placed in scintillant), or inthe supernatant after the complexes are subsequently dissociated.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of TI-binding protein found in thebead fraction quantitated from the gel using standard electrophoretictechniques such as described in the appended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either TI or itscognate binding protein can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated TI molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with TI butwhich do not interfere with binding of upstream or downstream elementscan be derivatized to the wells of the plate, and TI trapped in thewells by antibody conjugation. As above, preparations of a Ti-bindingprotein and a test compound are incubated in the TI-presenting wells ofthe plate, and the amount of complex trapped in the well can bequantitated. Exemplary methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the TIbinding element, or which are reactive with TI protein and compete withthe binding element; as well as enzyme-linked assays which rely ondetecting an enzymatic activity associated with the binding element,either intrinsic or extrinsic activity. In the instance of the latter,the enzyme can be chemically conjugated or provided as a fusion proteinwith the TI binding protein. To illustrate, the TI binding protein canbe chemically cross-linked or genetically fused with horseradishperoxidase, and the amount of polypeptide trapped in the complex can beassessed with a chromogenic substrate of the enzyme, e.g.3,3'-diamino-benzadine tetrahydrochloride or 4-chloro-1-napthol.Likewise, a fusion protein comprising the polypeptide andglutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J. Biol. Chem.249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-TI antibodies, can be used. Alternatively, the protein to bedetected in the complex can be "epitope tagged" in the form of a fusionprotein which includes, in addition to the TI sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include mycepitopes(e.g., see Ellison et al. (1991) J. Biol. Chem. 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharmacia, N.J.).

Cell Based Assays

In addition to cell-free assays, such as described above, the readilyavailable TI proteins provided by the present invention also facilitatesthe generation of cell-based assays for identifying small moleculeagonists/antagonists and the like. For example, cells which aresensitive to apoptosis can be caused to overexpress a recombinant TIprotein in the presence and absence of a test agent of interest, withthe assay scoring for modulation in TI responses by the target cellmediated by the test agent. As with the cell-free assays, agents whichproduce a statistically significant change in TI-dependent responses(either inhibition or potentiation) can be identified. In anillustrative embodiment, the expression or activity of a TI is modulatedin embryos or cells and the effects of compounds of interest on thereadout of interest (such as apoptosis) are measured. For example, theexpression of genes which are up- or down-regulated in response to aTI-dependent signal cascade can be assayed. In preferred embodiments,the regulatory regions of such genes, e.g., the 5' flanking promoter andenhancer regions, are operably linked to a detectable marker (such asluciferase) which encodes a gene product that can be readily detected.

Further, the transgenic animals described herein may be used to generatecell lines, containing one or more cell types involved in a weightdisorder, that can be used as cell culture models for diseases ordisorders described herein. While primary cultures derived fromtransgenic animals of the invention may be utilized, the generation ofcontinuous cell lines is preferred. For examples of techniques which maybe used to derive a continuous cell line from the transgenic animals,see Small et al. (1985) Mol. Cell Biol. 5:642-648.

In the event that the TI proteins themselves, or in complexes with otherproteins, are capable of binding DNA and modifying transcription of agene, a transcriptional based assay could be used, for example, in whicha TI-responsive regulatory sequence is operably linked to a detectablemarker gene.

Monitoring the influence of compounds on cells may be applied not onlyin basic drug screening, but also in clinical trials. In such clinicaltrials, the expression of a panel of genes may be used as a "read out"of a particular drug's therapeutic effect.

In yet another aspect of the invention, the subject TI polypeptides canbe used to generate a "two hybrid" assay (see, for example, U.S. Pat.No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and PCT Publication No. WO 94/10300), for isolating (coding sequencesfor other cellular proteins which bind to or interact with TI, such asthe C-terminus of tub, and the like. Briefly, the two hybrid assayrelies on reconstituting in vivo a functional transcriptional activatorprotein from two separate fusion proteins. In particular, the methodmakes use of chimeric genes which express hybrid proteins. Toillustrate, a first hybrid gene comprises the coding sequence for aDNA-binding domain of a transcriptional activator fused in frame to thecoding sequence for a TI polypeptide. The second hybrid protein encodesa transcriptional activation domain fused in frame to a sample gene froma cDNA library. If the bait and sample hybrid proteins are able tointeract, e.g., form a TI-dependent complex, they bring into closeproximity the two domains of the transcriptional activator. Thisproximity is sufficient to cause transcription of a reporter gene whichis operably linked to a transcriptional regulatory site responsive tothe transcriptional activator, and expression of the reporter gene canbe detected and used to score for the interaction of the TI and sampleproteins. The use of the subject TI molecules in a three hybrid assaywhich allows for phosphorylation of the assay components, such as forexample by the inclusion of src, or the PDGF cytoplasmic domain is alsoprovided for.

Transgenic Animals

These systems may be used in a variety of applications. For example, thecell- and animal-based model systems may be used to further characterizeTI genes and proteins. In addition, such assays may be utilized as partof screening strategies designed to identify compounds which are capableof ameliorating disease symptoms. Thus, the animal- and cell-basedmodels may be used to identify drugs, pharmaceuticals, therapies andinterventions which may be effective in treating disease.

Animal-Based Systems

One aspect of the present invention concerns transgenic animals whichare comprised of cells (of that animal) which contain a transgene of thepresent invention and which preferably (though optionally) express anexogenous TI protein in one or more cells in the animal. A TI transgenecan encode the wild-type form of the protein, or can encode homologuesthereof including both agonists and antagonists, as well as antisenseconstructs. In preferred embodiments, the expression of the transgene isrestricted to specific subsets of cells, tissues or developmental stagesutilizing, for example, cis-acting sequences that control expression inthe desired pattern. In the present invention, such mosaic expression ofa TI protein can be essential for many forms of lineage analysis and canadditionally provide a means to assess the effects of, for example, lackof TI expression which might grossly alter development in small patchesof tissue within an otherwise normal embryo. Toward this and,tissue-specific regulatory sequences and conditional regulatorysequences can be used to control expression of the transgene in certainspatial patterns. Moreover, temporal patterns of expression can beprovided by, for example, conditional recombination systems orprokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase"target sequence" refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of one of the subject TI proteins. For example, excision of atarget sequence which interferes with the expression of a recombinant TIgene, such as one which encodes an antagonistic homologue or anantisense transcript, can be designed to activate expression of thatgene. This interference with expression of the protein can result from avariety of mechanisms, such as spatial separation of the TI gene fromthe promoter element or an internal stop codon. Moreover, the transgenecan be made wherein the coding sequence of the gene is flanked byrecombinase recognition sequences and is initially transfected intocells in a 3' to 5' orientation with respect to the promoter element. Insuch an instance, inversion of the target sequence will reorient thesubject gene by placing the 5' end of the coding sequence in anorientation with respect to the promoter element which allow forpromoter driven transcriptional activation.

The transgenic animals of the present invention all include within aplurality of their cells a transgene of the present invention, whichtransgene alters the phenotype of the "host cell" with respect toregulation of cell growth, death and/or differentiation. Since it ispossible to produce transgenic organisms oi the invention utilizing oneor more of the transgene constructs described herein, a generaldescription will be given of the production of transgenic organisms byreferring generally to exogenous genetic material. This generaldescription can be adapted by those skilled in the art in order toincorporate specific transgene sequences into organisms utilizing themethods and materials described below.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) Proc. Natl. Acad. Sci. USA89:6232-6236; Orban et al. (1992) Proc. Natl. Acad. Sci. USA89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al. (1991) Science 251:1351-1355; PCT Publication No. WO92/15694) can be used to generate in vivo site-specific geneticrecombination systems. Cre recombinase catalyzes the site-specificrecombination of an intervening target sequence located between loxPsequences. loxP sequences are 34 base pair nucleotide repeat sequencesto which the Cre recombinase binds and are required for Cre recombinasemediated genetic recombination. The orientation of loxP sequencesdetermines whether the intervening target sequence is excised orinverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of a recombinant TI protein can be regulatedvia control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant TI protein requires the construction of a transgenic animalcontaining transgenes encoding both the Cre recombinase and the subjectprotein. Animals containing both the Cre recombinase and a recombinantTI gene can be provided through the construction of "double" transgenicanimals. A convenient method for providing such animals is to mate twotransgenic animals each containing a transgene, e.g., a TI gene andrecombinase gene.

One advantage derived from initially constructing transgenic animalscontaining a TI transgene in a recombinase-mediated expressible formatderives from the likelihood that the subject protein, whether agonisticor antagonistic, can be deleterious upon expression in the transgenicanimal. In such an instance, a founder population, in which the subjecttransgene is silent in all tissues, can be propagated and maintained.Individuals of this founder population can be crossed with animalsexpressing the recombinase in, for example, one or more tissues and/or adesired temporal pattern. Thus, the creation of a founder population inwhich, for example, an antagonistic TI transgene is silent will allowthe study of progeny from that founder in which disruption of TImediated induction in a particular tissue or at certain developmentalstages would result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the TI transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the trans-activatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, a TI transgene could remain silent intoadulthood until "turned on" by the introduction of the trans-activator.

In an exemplary embodiment, the "transgenic non-human animals" of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-2^(q) haplotypes such as C57BL/6 or DBA/1. Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or (completely suppressed) .

In one embodiment, the transgene construct is introduced into a singlestage embryo. The zygote is the best target for micro-injection. In themouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2pl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442). As a consequence,all cells of the transgenic animal will carry the incorporatedtransgene. This will in general also be reflected in the efficienttransmission of the transgene to offspring of the founder since 50% ofthe germ cells will harbor the transgene.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear. At about this time, the nucleotide sequence comprisingthe transgene is introduced into the female or male pronucleus asdescribed below. In some species such as mice, the male pronucleus ispreferred. It is most preferred that the exogenous genetic material beadded to the male DNA complement of the zygote prior to its beingprocessed by the ovum nucleus or the zygote female pronucleus. It isthought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote.

Thus, it is preferred that the exogenous genetic material be added tothe male complement of DNA or any other complement of DNA prior to itsbeing affected by the female pronucleus. For example, the exogencusgenetic material is added to the early male pronucleus, as soon aspossible after the formation of the male pronucleus, which is when themale and female pronuclei are well separated and both are located closeto the cell membrane. Alternatively, the exogenous genetic materialcould be added to the nucleus of the sperm after it has been induced toundergo decondensation. Sperm containing the exogenous genetic materialcan then be added to the ovum or the decondensed sperm could be added tothe ovum with the transgene constructs being added as soon as possiblethereafter.

Introduction of the transgene nucleotide sequence into the embryo may beaccomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gametc, or gametes. Thus, the gamete nuclei mustbe ones which are naturally compatible, i.e., ones which result in aviable zygote capable of undergoing differentiation and developing intoa functioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more that one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material which canbe added to the nucleus of the zygote or to the genetic material whichforms a part of the zygote nucleus. If no genetic material is removed,then the amount of exogenous genetic material which can be added islimited by the amount which will be absorbed without being physicallydisruptive. Generally, the volume of exogenous genetic material insertedwill not exceed about 10 picoliters. The physical effects of additionmust not be so great as to physically destroy the viability of thezygote. The biological limit of the number and variety of DNA sequenceswill vary depending upon the particular zygote and functions of theexogenous genetic material and will be readily apparent to one skilledin the art, because the genetic material, including the exogenousgenetic material, of the resulting zygote must be biologically capableof initiating and maintaining the differentiation and development of thezygote into a functional organism.

The number of copies of the transgene constructs which are added to thezygote is dependent upon the total amount of exogencus genetic materialadded and will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct, in order to insure that one copy is functional. Asregards the present invention, there will often be an advantage tohaving more than one functioning copy of each of the inserted exogenousDNA sequences to enhance the phenotypic expression of the exogenous DNAsequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and is used inthe art.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

The transgenic animals produced in accordance with the present inventionwill include exogenous genetic material. As set out above, the exogenousgenetic material will, in certain embodiments, be a DNA sequence whichresults in the production of a TI protein (either agonistic orantagonistic), and antisense transcript, or a TI mutant. Further, insuch embodiments the sequence will be attached to a transcriptionalcontrol element, e.g., a promoter, which preferably allows theexpression of the transgene product in a specific type of cell.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad.Sci USA 73:1260-1264). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida(Manipulating the Mouse Embryo, Hogan eds. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1986). The viral vector systemused to introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (Jahner et al. (1 985) Proc. Natl.Acad. Sci. 82:6927-6931; Van der Putten et al. (1985) Proc. Natl. Acad.Sci. USA 82:6148-6152). Transfection is easily and efficiently obtainedby culturing the blastomeres on a monolayer of virus-producing cells(Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298: 623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs; only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infectior of the midgestation embryo(Jahner et al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implani:ation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) Proc. Natl. Acad. Sci. USA 83:9065-9069; and Robertson et al.(1986) Nature 322:445-448). Transgenes can be efficiently introducedinto the ES cells by DNA transfection or by retrovirus-mediatedtransduction. Such transformed ES cells can thereafter be combined withblastocysts from a non-human animal. The ES cells thereafter colonizethe embryo and contribute to the germ line of the resulting chimericanimal. For review see Jaenisch, R. (1988) Science 240:1468-1474.

In one embodiment, gene targeting, which is a method of using homologousrecombination to modify an animal's genome. can be used to introducechanges into cultured embryonic stem cells. By targeting a TI gene ofinterest in ES cells, these changes can be introduced into the germlinesof animals to generate chimeras. The gene targeting procedure isaccomplished by introducing into tissue culture cells a DNA targetingconstruct that includes a segment homologous to a target TI locus, andwhich also includes an intended sequence modification to the TI genomicsequence (e.g., insertion, deletion, point mutation). The treated cellsare then screened for accurate targeting to identify and isolate thosewhich have been properly targeted.

Gene targeting in embryonic stem cells is in fact a scheme contemplatedby the present invention as a means for disrupting a TI gene functionthrough the use of a targeting transgene construct designed to undergohomologous recombination with one or more TI genomic sequences. Thetargeting constrict can be arranged so that, upon recombination with anelement of a TI gene, a positive selection marker is inserted into (orreplaces) coding sequences of the targeted gene. The inserted sequencefunctionally disrupts the TI gene, while also providing a positiveselection trait. Exemplary TI targeting constructs are described in moredetail below.

Generally, the embryonic stem cells (ES cells) used to produce theknockout animals will be of the same species as the knockout animal tobe generated. Thus for example, mouse embryonic stem cells will usuallybe used for generation of knockout mice.

Embryonic stem cells are generated and maintained using methods wellknown to the skilled artisan such as those described by Doetschman etal. (1985) J. Embryol. Exp. Morphol. 87:27-45). Any line of ES cells canbe used, however, the line chosen is typically selected for the abilityof the cells to integrate into and become part of the germ line of adeveloping embryo so as to create germ line transmission of the knockoutconstruct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog no. CKL 1934) Still another preferred ES cell line is the WW6cell line (Ioffe et al. (1995) Proc. Natl. Acad. Sci. USA 92:7357-7361).The cells are cultured and prepared for knockout construct insertionusing methods well known to the skilled artisan, such as those set forthby Robertson in: Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. IRL Press, Washington, D.C., 1987); byBradley et al. (1986) Current Topics in Devel. Biol. 20:357-371); and byHogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Insertion of the knockout construct into the ES cells can beaccomplished using a variety of methods well known in the art includingfor example, electroporation, microinjection, and calcium phosphatetreatment. A preferred method of insertion is electroporation.

Each knockout construct to be inserted into the cell must first be inthe linear form. Therefore, if the knockout construct has beer insertedinto a vector (described infra), linearization is accomplished bydigesting the DNA with a suitable restriction endonuclease selected tocut only within the vector sequence and not within the knockoutconstruct sequence.

For insertion, the knockout construct is added to the ES cells underappropriate conditions for the insertion method chosen, as is known tothe skilled artisan. Where more than one construct is to be introducedinto the ES cell, each knockout construct can be introducedsimultaneously or one at a time.

If the ES cells are to be electroporated, the ES cells and knockoutconstruct DNA are exposed to an electric pulse using an electroporationmachine and following the manufacturer's guidelines for use. Afterelectroporation, the ES cells are typically allowed to recover undersuitable incubation conditions. The cells are then screened for thepresence of the knockout construct.

Screening can be accomplished using a variety of methods. Where themarker gene is an antibiotic resistance gene, for example, the ES cellsmay be cultured in the presence of an otherwise lethal concentration ofantibiotic. Those ES cells that survive have presumably integrated theknockout construct. If the marker gene is other than an antibioticresistance gene, a Southern blot of the ES cell genom DNA can be probedwith a sequence of DNA designed to hybridize only to the markersequence. Alternatively, PCR can be used. Finally, if the marker gene isa gene that encodes an enzyme whose activity can be detected (e.g.,b-galactosidase), the enzyme substrate can be added to the cells undersuitable conditions, and the enzymatic activity can be analyzed. Oneskilled in the art will be familiar with other useful markers and themeans for detecting their presence in a given cell. All such markers arecontemplated as being included within the scope of the teaching of thisinvention.

The knockout construct may integrate into several locations in the EScell genome, and may integrate into a different location in each EScell's genome due to the occurrence of random insertion events. Thedesired location of insertion is in a complementary position to the DNAsequence to be knocked out, e.g., the TI coding sequence,transcriptional regulatory sequence, etc. Typically, less than about1-5% of the ES cells that take up the knockout construct will actuallyintegrate the knockout construct in the desired location. To identifythose ES cells with proper integration of the knockout construct, totalDNA can be extracted from the ES cells using standard methods. The DNAcan then be probed on a Southern blot with a probe or probes designed tohybridize in a specific pattern to genomic DNA digested with particularrestriction enzyme(s). Alternatively, or additionally, the genomic DNAcan be amplified by PCR with probes specifically designed to amplify DNAfragments of a particular size and sequence (i.e., only those cellscontaining the knockout construct in the proper position will generateDNA fragments of the proper size).

After suitable ES cells containing the knockout construct in the properlocation have been identified, the cells can be inserted into an embryo.Insertion may be accomplished in a variety of ways known to the skilledartisan, however a preferred method is by microinjection. Formicroinjection, about 10-30 cells are collected into a micropipette andinjected into embryos that are at the proper stage of development topermit integration of the foreign ES cell containing the knockoutconstruct into the developing embryo. For instance, as the appendedExamples describe, the transformed ES cells can be microinjected intoblastocysts.

The suitable stage of development for the embryo used for insertion ofES cells is very species dependent, however for mice it is about 3.5days. The embryos are obtained by perfusing the uterus of pregnantfemales. Suitable methods for accomplishing this are known to theskilled artisan, and are set forth by, e.g., Bradley et al. (supra).

While any embryo of the right stage of development is suitable for use,preferred embryos are male. In mice, the preferred embryos also havegenes coding for a coat color that is different from the coat colorencoded by the ES cell genes. In this way, the offspring can be screenedeasily for the presence of the knockout construct by looking for mosaiccoat color (indicating that the ES cell was incorporated into thedeveloping embryo). Thus, for example, if the ES cell line carries thegenes for white fur, the embryo selected will carry genes for black orbrown fur.

After the ES cell has been introduced into the embryo, the embryo may beimplanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent. For mice, this stage is about2-3 days pseudopregnant.

Offspring that are born to the foster mother may be screened initiallyfor mosaic coat color where the coat color selection strategy (asdescribed above, and in the appended examples) has been employed. Inaddition, or as an alternative, DNA from tail tissue of the offspringmay be screened for the presence of the knockout construct usingSouthern blots and/or PCR as described above. Offspring that appear tobe mosaics may then be crossed to each other, if they are believed tocarry the knockout construct in their germ line, in order to generatehomozygous knockout animals. Homozygotes may be identified by Southernblotting of equivalent amounts of genomic DNA from mice that are theproduct of this cross, as well as mice that are known heterozygotes andwild type mice.

Other means of identifying and characterizing the knockout offspring areavailable. For example, Northern blots can be used to probe the mRNA forthe presence or absence of transcripts encoding either the gene knockedout, the marker gene, or both. In addition, Western blots can be used toassess the level of expression of the TI gene knocked out in varioustissues of the offspring by probing the Western blot with an antibodyagainst the particular TI protein, or an antibody against the markergene product, where this gene is expressed. Finally, in situ analysis(such as fixing the cells and labeling with antibody) and/or FACS(fluorescence activated cell sorting) analysis of various cells from theoffspring can be conducted using suitable antibodies to look for thepresence or absence of the knockout construct gene product.

Yet other methods of making knock-out or disruption transgenic animalsare also generally known. See, for example, Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of a TI-gene can bccontrolled by recombinase sequences (described infra).

Animals containing more than one knockout construct and/or more than onetransgene expression construct are prepared in any of several ways. Thepreferred manner of preparation is to generate a series of mammals, eachcontaining one of the desired transgenic phenotypes. Such animals arebred together through a series of crosses, backcrosses and selections,to ultimately generate a single animal containing all desired knockoutconstructs and/or expression constructs, where the animal is otherwisecongenic (genetically identical) to the wild type except for thepresence of the knockout construct(s) and/or transgene(s) .

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. flames and S. J. Higgins eds.; Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES Identification of Tub Interactors

The following materials and methods were used in the Examples:

Yeast strains, Media, and Microbiological Techniques

Standard yeast media including synthetic complete medium lackingL-leucine, L-tryptophan, and L-histidine were prepared and yeast geneticmanipulations were performed as described (Sherman (1991) Meth. Enzymol.194:3-21). Yeast transformations were performed using standard protocols(Gietz et al. (1992) Nucl. Acids Res. 20:1425. Ito et al, (1983) J.Bacteriol. 153:163-168). Plasmid DNAs were isolated from yeast strainsby a standard method (Hoffman and Winston (1987) Gene 57:267-272).

Western Blotting

A total protein extract of TB14 and TB20 was subjected to Westernblotting analysis to confirm and qualitatively evaluate expression ofthe GAL4 DNA-binding domain TUB fusion proteins. The protein cataractwere prepared by growing TB14 and TB20 in synthetic complete mediumlacking L-tryptophan (Sherman (1991) Meth. Enzymol. 194:3) to an OD₆₀₀of 1. The yeast cells from 4.5 ml of culture were collected bycentrifugation and the cell pellet was resuspended in 1 ml of 0.25 MNaOH 1% beta-mercaptoethanol and incubated at 4° C. for 10 minutes. 160ml of 50% TCA were then added to the cell suspension and after mixingthe suspension was incubated at 4° C. for 10 minutes. The suspension wasthen microfuged at 4° C. for 10 minutes, the supernatant fraction wasdiscarded, and the pellet was washed with cold acetone, air dried, andthen resuspended in 120 ml of 2× tris-glycine SDS sample buffer (Novex,San Diego, Calif.) diluted to 1× strength with deionized water.

15 μl of the sample was boiled for 2 minutes and then electrophoresed ona 14% tris glycine SDS polyacrylamide gel (Novex) and then transferredto an immobilon PVDF membrane (Millipore; San Francisco, Calif.). Theprimary antibody utilized was a rabbit anti-yeast GAL4 DNA-bindingdomain polyclonal antibody (Upstate Biotechnology Inc., Lake Placid,N.Y.) and the secondary antibody was a donkey anti-rabbit Ig, peroxidaselinked species-specific whole antibody (Amersham Life Sciences,Cleveland, Ohio). Western blotting procedures were essentially asdescribed (Sambrook et al. Molecular Cloning 2nd edition. Cold SpringHarbor Laboratory Press. 1989) and proteins interacting with theantibodies were visualized using the ECL detection system (Amersham LifeSciences, Cleveland, Ohio), essentially as described by themanufacturer. Expression of the GAL4 DNA-binding domain TUB cytoplasmicdomain fusion proteins were detected.

Beta Galactosidase Assays

The filter disk beta-galactosidase (beta-gal) assay was performedessentially as previously described (Brill et al. (1994) Mol. Biol.Cell. 5:297-312). Briefly, strains to be tested were grown as patches ofcells on appropriate medium dictated by the experiment at 30° C.overnight. The patches or colonies of cells were replica plated toWhatman #50 paper disks (Schleicher & Schuell, #576; Keene, N.H.) thathad been placed on the test medium in petri dishes. After growthovernight at 30° C., the paper disks were removed from the plates andthe cells on them were permeabilized by immediately immersing them inliquid nitrogen for 30 seconds. After this treatment, the paper diskswere thawed at room temperature for 20 seconds and then placed in petridishes that contained a disk of Whatman #3 paper (Schleicher & Schuell,#593, Keene, N.H.) saturated with 2.5 ml of Z buffer containing 37 μl of2% weight per volume of the chromogenic beta-gal substrate X-gal. Thepermeabilized strains on the paper disks were incubated at 30° C. andinspected at timed intervals for the blue color diagnostic of beta-galactivity in this assay. The assay was stopped by removing the paper diskcontaining the patches of cells and air drying it.

Two Hybrid Screening and Identification of Tub Interactors

Human TUB 184-506 and human TUB 1-506 were cloned into pGBT9 (Clontech,Palo Alto, Calif.). The human TUB 184-506 was called pGBhTUB and thehuman TUB 1-506 clone was called pMB71. pGBhTUB and pMB71 weretransformed into two-hybrid screening strain HF7c. A pGBhTUBtransformant was called TB 14 and a pMB71 transformant was called TB20.It was verified that neither human TUB 184-506 nor human TUB 1-506activated the HIS3 or lacZ reporler genes present in HF7c. Proteinextracts from TB14 and TB20 were subjected to Western blot analysis.Human TUB 184-506 was expressed at a high level and human TUB 1-506 wasexpressed at a very low level.

In one experiment, TB14 was transformed with a human prostate two-hybridlibrary and 20 million transformants were obtained and in anotherexperiment TB14 was transformed with a mouse T-cell library and 10million transformants were obtained. TB20 was transformed with a humanprostate two-hybrid library and 1.5 million transformants were obtained.By PCR with TUB-specific primers, it was determined that the TUB cDNAwas present in these libraries. Transformants were plated on syntheticcomplete medium lacking leucine, tryptophan, and histidine to select fortransformants expressing cDNA library plasmids encoding TUB-interactingproteins. All colonies that grew on the selective plates were analyzedfor beta-galactosidase expression using the filter beta-galactosidaseassay and the strongest beta-galactosidase expressing plasmids from eachscreen were analyzed.

In the screen where TB14 was transformed with a human prostatetwo-hybrid library, E. coli plasmid ptyhq058; E. coli plasmid ptyhq054and E. coli plasmid ptyhq036 were identified. In the screen where TB 14was transformed with a mouse T-cell library, E. coli plasmid ptyht101and E. coli plasmid ptyht102, the mouse homologues of E. coli plasmidptyhq036 and E. coli plasmid ptyhq054 were identified. In the screenwhere TB20 was transformed with the human prostate library, E. coliplasmid ptyhq049 and human serine palmitoyl transferase (GenBankAccession No. U15555) were identified. Human serine palmitoyltransferase is a weak interactor because it activates the HIS3 reportergene but not the lacZ gene, at least not enough to be detected in theassays. E. coli plasmid ptyhq058 appeared to be the strongestinteractor. All seven of these interactors bind to full length human andmouse TUB and the carboxyl-terminus of human and mouse TUB. In addition,none of these interactors bind to the carboxyl-terminus of human andmouse TUB missing the final 44 amino acids, amino acids lacking in themutated mouse TUB gene. These seven interactors were found to not bindto several test proteins showing that they bind specifically to TUB.

Northern Analysis

Methods

Total RNA was isolated from various mouse (C57BL/6 wild type andtub/tub) tissues using RNAzol B (Tel-Test, Inc., Friendswood, Tex.).Poly A+RNA was isolated from a variety of human and mouse cell linesusing the FastTrack system (Invitrogen, San Diego, Calif.). ExtractedRNA was electrophoresed through a formaldehyde gel, transferred toGenescreen nylon membrane (NEN Research Products, Boston, Mass.) andcross-liked using a Stratalinker apparatus (Stratagene, La Jolla,Calif.).

For probing northern blots, 50 ng of the following probes were labelledusing Prime-It (Stratagene, La Jolla, Calif.): human ank; human tpr;human ring; mouse tpr; or mouse ring. Blots were hybridized at 65° C. inChurch Buffer overnight and washed in 0.2× SSC/0.1% SDS also at 65° C.Filters were exposed to film (X-omat AR, Kodak) for 18-36 hours.

Human Tissue Results

Human multiple tissue northern blots (Clontech, Palo Alto, Calif.) wereprobed. The human tissues tested included: spleen, thymus, prostate,testes, uterus, small intestine, colon, peripheral blood leukocytes,heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreasand retina.

Bands of approximately 2.4 kb and 10 kb were found to be ubiquitouslyexpressed in all tissues tested using the human ank probe. The 2.4 kbband in retinal tissue gave an increased signal. Using the human ringprobe, bands of 1.3 kb and 2 kb were expressed in all tissues tested.The 2 kb band gave an increased signal in retinal tissue. Hybridizationwith the human ring probe yielded bands of 3 kb and 4 kb in all tissuestested. An additional band of 1.4 kb was detected in tests.

Mouse Tissue Results

Mouse tissues were obtained from C57BL/6 and tub/tub animals. Tissuesused were: brain, hypothalamus, liver, heart, spleen, stomach, kidney,muscle, fat, and testes. Neither the human ring probe nor the human ankprobe yielded any signal in any tissue tested. the mouse tpr probehybridized with a 1.4 kb band in C57BL/6 testes and a 1.4 kb band intub/tub brain and testes. The mouse ring probe hybridized with a 2.4 kband a 3.0 kb band in all tissues tested from both strains of mice andalso hybridized with a 1.4 kb band from testes tissue from C57BL/6 andtub/tub mice.

Ceil Line Results

Poly A+RNA was isolated from a variety of ATCC cell lines (includinghuman cell lines SHEP; SHSY5Y; SKNMC (neuroblistoma); SKNSH; Neuro 2A(neuroblastoma), NB412A/8; the human breast carcinoma cell line MCF7 andthe mouse fibroblast cell line NIH 3T3). The human ank probe hybridizedwith a 2.3 kb band in the SHEP, SHSY5Y, SKNMC, SKNSH, and MCF7 celllines. The same human ank probe lit up a 2 kb band in Neuro 2A andNB412A/8 cells. No signal was detected in the 3T3 cell line. The humantpr probe hybridized with a 2 kb band in all cell lines tested. Anadditional band of 4.4 kb was detected using this probe in the neuro 2Acells. The human ring probe detected a 2.4 kb band in the SHEP, SHSY5Y,SKNMC, and SKNSH cell lines. No signal was detected in any other of thecell lines using the ring probe.

Deposit of Microorganisms

E. coli plasmid ptyhq049 was deposited with the American Type CultureCollection 10801 University Boulevard, Manassas, Va. 20110-2209 on Aug.6, 1996 under the terms of the Budapest Treaty and assigned AccessionNumber 98125 (hTI-1) (SEQ ID NO:1).

E. coli plasmid ptyhq058 was deposited with the American Type CultureCollection 10801 University Boulevard, Manassas, Va. 20110-2209 on Aug.6, 1996 under the terms of the Budapest Treaty and assigned AccessionNumber 98127 (hTI-2) (SEQ ID NO:2).

E. coli plasmid ptyhq036 was deposited with the American Type CultureCollection 10801 University Boulevard, Manassas, Va. 20110-2209 on Aug.6, 1996 under the terms of the Budapest Treaty and assigned AccessionNumber 98128 (hTI-3) (SEQ ID NO:3).

E. coli plasmid ptyhq054 was deposited with the American Type CultureCollection 10801 University Boulevard, Manassas, Va. 20110-2209 on Aug.6, 1996 under the terms of the Budapest Treaty and assigned AccessionNumber 98126 (hTI-4) (SEQ ID NO:5).

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 36    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1386 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - GAATTCGGCA CGAGCGCACT CGCAGCCCTG GCAGGCGGCA CTGGTCATGG AA - #AACGAATT      60    - GTTCTGCTCG GGCGTCCTGG TGCATCCGCA GTGGGTGCTG TCAGCCGCAC AC - #TGTTTCCA     120    - GAAGTGAGTG CAGAGCTCCT ACACCATCGG GCTGGGCCTG CACAGTCTTG AG - #GCCGACCA     180    - AGAGCCAGGG AGCCAGATGG TGGAGGCCAG CCTCTCCGTA CGGCACCCAG AG - #TACAACAG     240    - ACCCTTGCTC GCTAACGACC TCATGCTCAT CAAGTTGGAC GAATCCGTGT CC - #GAGTCTGA     300    - CACCATCCGG AGCATCAGCA TTGCTTCGCA GTGCCCTACC GCGGGGAACT CT - #TGCCTCGT     360    - TTCTGGCTGG GGTCTGCTGG CGAACGGCAG AATGCCTACC GTGCTGCAGT GC - #GTGAACGT     420    - GTCGGTGGTG TCTGAGGAGG TCTGCAGTAA GCTCTATGAC CCGCTGTACC AC - #CCCAGCAT     480    - GTTCTGCGCC GGCGGAGGGC AAGACCAGAA GGACTCCTGC AACGGTGACT CT - #GGGGGGCC     540    - CCTGATCTGC AACGGGTACT TGCAGGGCCT TGTGTCTTTC GGAAAAGCCC CG - #TGTGGCCA     600    - AGTTGGCGTG CCAGGTGTCT ACACCAACCT CTGCAAATTC ACTGAGTGGA TA - #GAGAAAAC     660    - CGTACCAGGC CAGTTAACTC TGGGGACTGG GAACCCATGA AATTGACCCC CA - #AATACATC     720    - CTGCGGAAGG AATTCAGGAA TATCTGTTCC CAGCCCCTCC TCCCTCAGGC YC - #AGGAGTCC     780    - AGGCCCCCAG CCCCTCCTCC CTCAAACCAA GGGTACAGAT CCCCAGCCCC TC - #CTCCCTCA     840    - GACCCAGGAG TCCAGACCCC CCAGCCCCTC CTCCCTCAGA CCCAGGAGTC CA - #GCCCCTCC     900    - TCCCTCAGAC CCAGGAGTCC AGACCCCCCA GCCCCTCCTC CCTCAGACCC AG - #GGGTCCAG     960    - CCTCTCCTCC CTCAGACCCA GGAGTCCAGA CCCCCCAGCC CCTCCTCCCT CA - #GACCCAGG    1020    - AGTCCAGCCC CTCCTCCCTC AGACCCAGGA GTCCAGATCC CCCAGCCCCT CC - #TCCCTCAG    1080    - ACCCAGGGGT CCAGGCCCCC AACCCCTCCT CCCTCAGACT CAGAGGTCCA AG - #CCCCCAAC    1140    - CCCTCCTTCC CCAGACCCAG AGGTCCAGGT ACCAGCCCCT CCTCCCTCAG AC - #CCAGCGGT    1200    - CCAATGCCAC CTATACTCTC CCTGTACANA TTGCCNCCTT GTGGCACGTT GA - #CCCAACCT    1260    - TACCAGTTGG TTTTTCATTT TTTGTCCCTT TCCCCTAGAT CCAGAAATAA AG - #TTTAAGRG    1320    - RAGSGCCAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA - #AAAACYCG    1380    #         1386    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2103 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - GGATCCGAAT TCGGCACGAG GCGGAGGGAA GTAGGTCCGT TGGTCGGTCG GG - #AACGAGGC      60    - TCAGGCGGCC AGGCCCGCGC GGAGCCGTTG CCATGGCAGC CGCCGCCGGG GA - #CGCGGACG     120    - ACGAGCCGCG CTCAGGCCAC TCGAGCTCGG AGGGCGAGTG CGCGGTGGCG CC - #GGAGCCGC     180    - TGACTGACGC TGAGGGCCTC TTCTCCTTCG CTGACTTCGG GTCTGCGCTG GG - #CGGCGGCG     240    - GCGCGGGCCT CTCGGGCCGG GCGTCCGGCG GGGCCCAGTC GCCGCTGCGC TA - #CTTGCACG     300    - TCCTGTGGCA GCAGGATGCG GAGCCGCGCG ACGAGCTGCG CTGCAAGATA CC - #CGCTGGCC     360    - GGCTGAGGCG CGCTGCCAGG CCCCACCGGC GGCTCGGGCC CACGGGCAAG GA - #GGTGCACG     420    - CTCTGAAGAG ACTGAGGGAC TCGGCCAATG CCAATGATGT GGAAACAGTG CA - #GCAGCTGC     480    - TGGAAGATGG CGCGGATCCC TGTGCAGCTG ATGACAAGGG CCGCACAGCT CT - #ACACTTTG     540    - CCTCATGCAA TGGCAATGAC CAGATTGCTG CTCCTGGACC ATGGTGCTGA TC - #CTAACCAG     600    - CGAGATGGGC TGGGGAACAC GCCACTGCAC CTGGCGGCCT GCACCAACCA CG - #TTCCTGTC     660    - ATCACCACAC TGCTACGAGG AGGGGCCCGT GTAGATGCCC TGGACCGAGC TG - #GTCGCACA     720    - CCCCTGCACC TGGCCAAGTC AAAGCTGAAT ATCCTGCAGG AGGGCCATGC CC - #AGTGCCTA     780    - GAGGCTGTGC GTCTGGAGGT GAAGCAGATC ATCCATATGC TGAGGGAGTA TC - #TGGAGCGC     840    - CTAGGGCAAC ATGAGCAGCG AGAACGCCTG GATGACCTCT GCACCCGCCT GC - #AGATGACC     900    - AGTACCAAAG AGCAGGTGGA TGAAGTGACT GACCTCCTGG CCAGCTTCAC CT - #CCCTCAGT     960    - CTGCAGATGC AGAGCATGGA GAAGAGGTAG CAAGAGAGGC TCCCTGCCTT CC - #TGCCACTG    1020    - CCCCACCCTG CCCCACTGCT GTCTCAGTAC CAAGAAAAAG CCCAACATCT GG - #GACTTGGA    1080    - GCTGCACTTG TCTGGTGAGG ACCTTGCCCT CACCCGCACA TGCCGTGGGG CA - #GAGATGCT    1140    - CTCTCTCCAC GGCCTCAGAG CCACTCCCAG CCACAGTTTC CAGCATCTCT GT - #GGACAGGG    1200    - ACCACAGCTC CCAGCTTCTT CCAGTTCTCG CAGCACCAGA CCAGCCTCTG CA - #GCTGCACT    1260    - TCAGCTCCGC AGACCTGCGC TATCTCAGCA GACCTCACTT GCCCCATGGC CT - #TCATGGCG    1320    - CGCTCCAGGC CTCAGACCCT TCTCTGTGTT CCGTCCTGGC CATGGGCTTG TT - #GCAGTCAG    1380    - CAGGTGTGGG CTTAGGCGGG CACCCTGTGG CCAGGGGTAC TGCGTGAGGC CC - #TCAGTTGG    1440    - TCCTGTGCCT CTCACCAGCA CTTAGACAGA CACGTCACCA GACTTTCAAG GA - #GATACTGC    1500    - AGTGAGTTTC TCTGGTTGGA AGGGGAGGGT TGGTGAGTCC CAGACCTTAA AA - #ATACAAGG    1560    - TTAAGAGGGA CCCCAAAGCA AAAAATTCCA ACCCTTTTCC TCCCAGTCAT TG - #AAACACCA    1620    - AAACTATTAT ACCGGAGGGT GTAATAGTTT TGCTGCCCAG TTGTGGTAGG CC - #AGTAGTGG    1680    - CCTCCCAAGA TGCCCATGTC CTAATCCCAG GAACCTGTCA AAATTACCTT GT - #ATGGCCAA    1740    - AGGGGCTTTG CAGATGTAAT GAAGTTAAGG ATCTTTCGCC AGGAAGATTA TC - #CCAGCTTG    1800    - TTCAGGAGGG CTTGATGTCC TCACCCGGGT CTGTATAACA GAAGAGCAGG TG - #ACGGGAGA    1860    - GGAGGTTGGA GGTGTAGCGA TGGAGCAGGA AACTGGAGTT GAGGAGGGCA GC - #TCAAGCCA    1920    - CAGAGTCCAG GCCACCTCAG AGCCAGGAAA TGCATCCTCC CACAGAGCCC TG - #GAAGGCCC    1980    - CAGCCCTGCT CCCACCTGGA CTGGCTCAGT GAGGCTAATT TTATAATTCT GG - #CTGATTTT    2040    - AGAACTCTAA GGGAATAAAT TTGTGTTGTT TTAAGTCAAA AAAAAAAAAA AA - #AAAAACTC    2100    #           2103    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1048 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - AATTCGGCAC GAGAAAAATG CTAGCTATTA TGGTAATCGA GCAGCCACCT TG - #ATGATGCT      60    - TGGAAGGTTC CGGGAAGCTC TTGGAGATGC ACAACAGTCA GTGAGGTTGG AT - #GACAGTTT     120    - TGTCCGGGGA CATCTACGAG AGGGCAAGTG CCACCTCTCT CTGGGGAATG CC - #ATGGCAGC     180    - ATGTCGCAGC TTCCAGAGAG CCCTAGAACT GGATCATAAA AATGCTCAGG CA - #CAACAAGA     240    - GTTCAAGAAT GCTAATGCAG TCATGGAATA TGAGAAAATA GCAGAAACAG AT - #TTTGAGAA     300    - GCGAGATTTT CGGAAGGTTG TTTTCTGCAT GGACCGTGCC CTAGAATTTG CC - #CCTGCCTG     360    - CCATCGCTTC AAAATCCTCA AGGCAGAATG TTTAGCAATG CTGGGTCGTT AT - #CCAGAAGC     420    - ACAGTCTGTG GCTAGTGACA TTCTACGAAT GGATTCCACC AATGCAGATG CT - #CTGTATGT     480    - ACGAGGTCTT TGCCTTTATT ACGAAGATTG TATTGAGAAG GCAGTTCAGT TT - #TTCGTACA     540    - GGCTCTCAGG ATGGCTCCTG ACCACGAGAA GGCCTGCATT GCCTGCAGAA AT - #GCCAAAGC     600    - ACTCAAAGCA AAGAAAGAAG ATGGGAATAA AGCATTTAAG GAAGGAAATT AC - #AAACTAGC     660    - ATATGAACTG TACACAGAAG CCCTGGGGAT AGACCCCAAC AATATAAAAA CA - #AATGCTAA     720    - ACTCTACTGT AATCGGGGTA CGGTTAATTC CAAGCTTAGG AAACTAGATG AT - #GCAATAGA     780    - AGACTGCACA AATGCAGTGA AGCTTGATGA CACTTACATA AAAGCCTACT TG - #AGAAGAGC     840    - TCAGTGTTAC ATGGACACAG AACAGTATGA AGAAGCAGTA CGAGACTATG AA - #AAAGTATA     900    - CCAGACAGAG AAAACAAAAG AACACAAACA GCTCCTAAAA AATGCGCAAC TT - #AAGTTTAG     960    - AAATTACAAG TTTCAGTAAT AGCTGAACCT GTTCAAAATG TTAATAAAGG TT - #TCGTTGCA    1020    #           1048   AAAA AAAAAAAA    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1700 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - TCGAGATTTA CCCATAGATA TGTGTCCTAA CAATGCCAGC TATTACGGTA AT - #CGAGCGGC      60    - CACACTGATG ATGCTTGGAC GGTTCCGGGA AGCTCTTGGA GATGCGCAGC AG - #TCTGTGAG     120    - GTTGGATGAC AGTTTTGTCC GGGGACACCT CCGAGAAGGC AAGTGCCACC TC - #TCACTTGG     180    - GAATGCAATG GCGGCATGTC GTAGTTTCCA AAGAGCCCTA GAACTGGATC AT - #AAAAATGC     240    - CCAGGCACAG CAGGAGTTCA AGAACGCCAA TGCCGTCATG GAGTATGAGA AA - #ATAGCAGA     300    - AGTGGATTTT GAAAAGCGAG ATTTCCGGAA GGTTGTTTTC TGCATGGACC GT - #GCCCTAGA     360    - ATTTGCCCCT GCCTGCCATC GATTCAAAAT TCTCAAAGCA GAATGTTTAG CA - #ATGCTTGG     420    - TCGATACCCA GAAGCACAGT TTGTGGCCAG TGACATTTTA CGAATGGATT CC - #ACCAATGC     480    - TGATGCTCTG TATGTCCGGG GTCTTTGCCT TTATTACGAA GATTGTATTG AG - #AAGGCAGT     540    - GCAGTTTTTT GTACAGGCTC TCAGGATGGC TCCTGACCAC GAGAAGGCTT GT - #GTCGCTTG     600    - TAGAAATGCC AAAGCCCTTA AAGCCAAGAA GGAAGATGGG AATAAAGCCT TT - #AAGGAAGG     660    - AAATTACAAG CTAGCATATG AACTGTACAC AGAAGCCTTG GGGATAGATC CC - #AACAACAT     720    - AAAAACAAAT GCTAAACTCT ACTGTAATCG GGGTACGGTT AATTCCAAGC TT - #AGGCAACT     780    - GGAAGATGCC ATAGAAGACT GTACAAATGC GGTGAAGCTC GATGACACTT AC - #ATCAAAGC     840    - CTACCTGAGA AGAGCTCAGT GTTACATGGA CACAGAGCAG TTTGAAGAAG CC - #GTGCGGGA     900    - CTATGAAAAA GTGTATCAGA CGGAGAAAAC AAAAGAACAC AAACAGCTCC TT - #AAGAATGC     960    - ACAGCTGGAA CTGAAGAAGA GCAAGAGGAA AGATTACTAC AAGATCCTGG GA - #GTGGACAA    1020    - GAATGCCTCT GAGGACGAGA TCAAGAAAGC TTACCGGAAA CGGGCCTTGA TG - #CACCATCC    1080    - AGATCGGCAC AGTGGGGCCA GTGCCGAAGT TCAGAAGGAG GAGGAGAAGA AG - #TTTAAGGA    1140    - AGTGGGAGAG GCCTTTACCA TCCTCTCTGA TCCCAAGAAA AAGACTCGTT AT - #GACAGTGG    1200    - ACAGGACTTG GATGAGGAGG GCATGAATAT GGGCGATTTT GATGCAAACA AC - #ATCTTCAA    1260    - GGCATTCTTC GGTGGTCCTG GGGGCTTCAG CTTTGAAGCA TCTGGCCCAG GG - #AATTTCTA    1320    - CTTTCAGTTT GGCTAATGAA GGCCAACTAC TTAAAACCCA GAAAATGCAG AC - #TTGCTTGG    1380    - TTTAACCATG AGTGTGGACA GTTCACTTCC TCCATCATGT CCCTGTGTAC TT - #ATAGCAGT    1440    - NTCGTTTTCT CAGTCGGGTG CCCTGTGTCT GTATGAGGGG TGAAKGAAAG GG - #GGCCAGTG    1500    - CTGAGGACTA GGGAGGGATG GAAGCCANGG GTAKACAGGG AAGCAGGCAG CT - #TGTGAATT    1560    - TTTGTTGTAT TGTTTAACTT TATTAAAAAA GAAAAACAAT ACTGTAAAWT WT - #AAAAAGGA    1620    - AAAGRATTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA - #AAAAAAAA    1680    #                 170 - #0    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1248 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - ACGAGCGGTG ACGGCCGGGT AGGCTGTAGG CAGCGCAATG CCAAGACAGA GC - #TGCTGGCG      60    - GCGGCGGGCG AATCTCCCTG CACCATGAGC CTCGGCTCCG GCCCCGTTAG GG - #GCCGATAA     120    - GCACAGCGCA CGCCGCCCTC CATTTGCCCC GGGGCCTCGG CTGCGAAGAT AG - #CGGCGGCC     180    - GGACAGGAAG CTCGAGGAAA GCGCTGGGCC GGGTCTCTAC GAACACGTGA AG - #GAAAAGCA     240    - GCTCCGTCCA CAACGCCGCT TCGGGGCTCC TAGGGAGTCG GGCCCCGGGC CG - #CCACCGTC     300    - ACCTCCGGCC GCTGCCGCTG TCGCCATCGC CTTGTTTCCC CATCCCCCGC CA - #TGGCCGAG     360    - GACCTCTCTG CGGCCACGTC CTACACCGAA GATGATTTCT ACTGCCCCGT CT - #GTCAGGAG     420    - GTGCTCAAAA CGCCCGTGCG GACCACGGCC TGTCAGCACG TTTTCTGTAG AA - #AATGTTTC     480    - CTGACTGCAA TGAGGGAAAG CGGAGCACAT TGTCCCCTAT GTCGTGGAAA TG - #TGACTAGA     540    - AGAGAGAGAG CATGTCCTGA ACGGGCCTTA GACCTTGAAA ATATAATGAG GA - #AGTTTTCT     600    - GGTAGCTGCA GATGCTGTGC AAAACAGATT AAATTCTATC GCATGAGACA TC - #ATTACAAA     660    - TCTTGTAAGA AGTATCAGGA TGAATATGGT GTTTCTTCTA TCATTCCAAA CT - #TTCAGATC     720    - TCTCAAGATT CAGTAGGGAA CAGCAATAGG AGTGAAACAT CCACATCTGA TA - #ACACAGAA     780    - ACTTACCAAG AGAATACAAG TTCTTCTGGT CATCCTACTT TTAAGTGTCC CC - #TGTGTCAA     840    - GAATCAAATT TTACCAGACA GCGTTTACTG GATCACTGTA ACAGTAATCA CC - #TATTTCAG     900    - ATAGTTCCTG TGACATGTCC TATTTGTGTG TCTCTTCCTT GGGGAGATCC TA - #GCCAGATT     960    - ACCAGAAATT TCGTTAGTCA TCTAAATCAG AGACATCAAT TTGATTATGG AG - #AATTTGTG    1020    - AATCTTCAGC TAGATGAAGA AACCCAATAC CAAACTGCTG TTGAAGAATT TT - #TTCAAGTA    1080    - AACATTTGAA GGCTGTAGAC ATTTTTGCAT TTTTGTACCT GCAAGTGCCA TC - #TTTAAGGG    1140    - GGAAAMTACA TGAAGTCACC GTTACAGTAA CTTGATGTGT ATATTAATAA AA - #GTAATTCA    1200    #              1248AAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2121 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - AGTTCACCTA CCACCACCAC CTCGGCTCCT GCCGGCGCCG TCGCCTCTCC CG - #CCCACCCC      60    - TCGCCATGTC CGAGGAACTT TCGGCGGCCA CGTCCTACAC GGAAGATGAT TT - #CTACTGCC     120    - CTGTCTGTCA GGAGGTGCTC AAGACGCCGG TGCGGACCGC GGCCTGTCAG CA - #CGTTTTCT     180    - GTAGAAAATG TTTCCTGACT GCAATGAGAG AAAGTGGAAT ACATTGTCCC CT - #ATGTCGTG     240    - GAAGTGTGAC TAGAAGAGAA AGAGCATGTC CGGAACGGGC CTTAGATCTT GA - #AAATATCA     300    - TGAGGAGGTT TTCTGGTAGC TGCAGATGCT GTTCAAAAAA GATTAAATTC TA - #TCGCATGA     360    - GACATCATTA CAAATCTTGT AAGAAGTATC AGGATGAATA TGGTGTTTCT TC - #TGTCATTC     420    - CAAACTTTAA GATTTCTCAA GATTCAGTAA GGAGCAGTAA TAGGAGTGAA AC - #ATCTGCAT     480    - CTGATAACAC AGAAACTTAT CAAGAGGATA CAAGTTCTTC TGGGCATCCT AC - #CTTTAAGT     540    - GTCCCTTATG TCAAGAGTCA AATTTCACCA GACAACGTTT ATTGGATCAC TG - #TAATAGTA     600    - ACCACCTATT TCAGATAGTT CCTGTGACAT GTCCTATTTG TGTGTCTCTT CC - #TTGGGGAG     660    - ATCCTAGCCA GATTACTAGA AATTTCGTTA GTCATCTAAA TCAAAGACAT CA - #GTTTGATT     720    - ATGGAGAATT TGTGAATCTT CAGCTAGATG AGGAAACCCA ATATCAAACT GC - #TGTGGAAG     780    - AGTCTTTTCA AGTAAACATG TGACATGTAT AGACATCTCT GCCTCCTTGC AA - #CCTACAAG     840    - TGCCATCTTT AAGGAGAAGA CATGAAGTCA CCATTTTCAG TAATTTGCTG TG - #CATATTAA     900    - TAAAAATAAT AATTCAGTCT ACTGTATTAG GTTTTTAATT GAAAATAAAG GT - #GGGCCACC     960    - CTAATACCAT TCTCTAGACA GTTACTTTAA CAGCATGGAA AGGGTTGTAT TT - #CACTTGTG    1020    - TGGTGAAAAG AGAATCTCTG TTGTCTTTTT CTTCCTTGTA TTACATATTC TC - #AATGTTTC    1080    - ATTAAGTTGT TTTTGGTATT TGATATAGTT CCTTCTGTTT AGTACAGAGA TA - #ACAGCAAA    1140    - TTCTGAACGA TGTGATTCTT AAAAAGCTAA TAAACCTGAG CCATTTGTCA GA - #GCTGTAGA    1200    - ATGGAAACTT GAAGTGTGAA GTGGGATAAT CCAAAGGGAT TTTTTTTTAA AG - #TATAGATT    1260    - CTAGCTGAGG AATTCAACAA TAAGAAAGTT GTATTTATGT AATGTTTAGT AT - #TTTTGAAG    1320    - ACTAGTGAGA TTTCTTTAAT AATTTTTACT TTGAAAGCAT ATTGTACAAA TG - #TTTCTTCT    1380    - TTTGCTATTA GAAGAACATC AAGAGAAGTT TCCTTTGGTG GTTAGTTTGT TA - #TTTCAATC    1440    - TAGGTTGAAT AATTTGTAAG CCTAAATGTT ATATACCACA GTTCTTTGTA GT - #CAGTATTT    1500    - CTCACTGGGT GATGAAACTT TTCAGCCAGT GAATGATACA TTCAATTAGT TT - #TTTAAAAA    1560    - TCCAAAGTTG CAGATGTATG TGGATATGTA CATAGACTTT TGCATGTATA TA - #TACACATA    1620    - TATATCTTTG CCTAGAGTTT GTCAGGTTAT GTATAGAATT TCTATTAAAA AG - #TTTTAATA    1680    - ATGGACAAGC AATATAGGAT TGAAGTATTT ATCTCCTTTG TTTAAAATTT TG - #TATGTTAC    1740    - CAAGTTTTTA AAACAGTAAG CCAAATACTA TGTGGTACAG TTGGCTGTTA TT - #ACACCTGA    1800    - AAAATGTTAA ATGGTGCTCA CTTGTTACGT TTGAAAATGA TGCATAACTG AC - #GTGTGGTG    1860    - AGAGATTTTA CCAGCTACTG TTTCACTACA TTTTAGTCAA AACAAAGTTT GT - #TCTTAATC    1920    - TTTGGTATAA AGTGTTGTAG AGAAGGCCAA GTCACAAAGT AAAGGGTGAA GG - #GGGAATTC    1980    - TGACATTCCA CACTAACATA ACACTGTTAT GCTTTCTTTA AAATAACTAA CC - #GCAAAAGA    2040    - AAATCTCTGA AGTAGTTTGC TGCTAATATA TACATATATT GTAAAAAAAA AA - #GGTATATT    2100    #                2121TC G    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #   17             C    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #       36         UCAC UAGAGCGAAA GCGGCA    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #   17             C    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #   17             G    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #  18              CC    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    #       36         UCAC UAGAGCGAAA AAUCUC    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #  18              CC    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #   17             A    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    #  18              GG    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    #       36         UCAC UAGAGCGAAA GGGCAC    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    #   17             G    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    #  18              CC    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 16 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    #    16    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    #       36         UCAC UAGAGCGAAA AGCAAU    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    #  18              GG    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 16 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    #    16    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    #  18              CC    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    #       36         UCAC UAGAGCGAAA UUCCAC    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    #   17             C    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    #  18              TC    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    #  18              GC    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    #       36         UCAC UAGAGCGAAA CCCGAA    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    #  18              GG    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    #  18              GG    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 232 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    - Asn Ser Ala Arg Ala His Ser Gln Pro Trp Gl - #n Ala Ala Leu Val Met    #                15    - Glu Asn Glu Leu Phe Cys Ser Gly Val Leu Va - #l His Pro Gln Trp Val    #            30    - Leu Ser Ala Ala His Cys Phe Gln Lys Xaa Va - #l Gln Ser Ser Tyr Thr    #        45    - Ile Gly Leu Gly Leu His Ser Leu Glu Ala As - #p Gln Glu Pro Gly Ser    #    60    - Gln Met Val Glu Ala Ser Leu Ser Val Arg Hi - #s Pro Glu Tyr Asn Arg    #80    - Pro Leu Leu Ala Asn Asp Leu Met Leu Ile Ly - #s Leu Asp Glu Ser Val    #                95    - Ser Glu Ser Asp Thr Ile Arg Ser Ile Ser Il - #e Ala Ser Gln Cys Pro    #           110    - Thr Ala Gly Asn Ser Cys Leu Val Ser Gly Tr - #p Gly Leu Leu Ala Asn    #       125    - Gly Arg Met Pro Thr Val Leu Gln Cys Val As - #n Val Ser Val Val Ser    #   140    - Glu Glu Val Cys Ser Lys Leu Tyr Asp Pro Le - #u Tyr His Pro Ser Met    145                 1 - #50                 1 - #55                 1 -    #60    - Phe Cys Ala Gly Gly Gly Gln Asp Gln Lys As - #p Ser Cys Asn Gly Asp    #               175    - Ser Gly Gly Pro Leu Ile Cys Asn Gly Tyr Le - #u Gln Gly Leu Val Ser    #           190    - Phe Gly Lys Ala Pro Cys Gly Gln Val Gly Va - #l Pro Gly Val Tyr Thr    #       205    - Asn Leu Cys Lys Phe Thr Glu Trp Ile Glu Ly - #s Thr Val Pro Gly Gln    #   220    - Leu Thr Leu Gly Thr Gly Asn Pro    225                 2 - #30    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 300 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    - Met Ala Ala Ala Ala Gly Asp Ala Asp Asp Gl - #u Pro Arg Ser Gly His    #                15    - Ser Ser Ser Glu Gly Glu Cys Ala Val Ala Pr - #o Glu Pro Leu Thr Asp    #            30    - Ala Glu Gly Leu Phe Ser Phe Ala Asp Phe Gl - #y Ser Ala Leu Gly Gly    #        45    - Gly Gly Ala Gly Leu Ser Gly Arg Ala Ser Gl - #y Gly Ala Gln Ser Pro    #    60    - Leu Arg Tyr Leu His Val Leu Trp Gln Gln As - #p Ala Glu Pro Arg Asp    #80    - Glu Leu Arg Cys Lys Ile Pro Ala Gly Arg Le - #u Arg Arg Ala Ala Arg    #                95    - Pro His Arg Arg Leu Gly Pro Thr Gly Lys Gl - #u Val His Ala Leu Lys    #           110    - Arg Leu Arg Asp Ser Ala Asn Ala Asn Asp Va - #l Glu Thr Val Gln Gln    #       125    - Leu Leu Glu Asp Gly Ala Asp Pro Cys Ala Al - #a Asp Asp Lys Gly Arg    #   140    - Thr Ala Leu His Phe Ala Ser Cys Asn Gly As - #n Asp Gln Ile Val Gln    145                 1 - #50                 1 - #55                 1 -    #60    - Leu Leu Leu Asp His Gly Ala Asp Pro Asn Gl - #n Arg Asp Gly Leu Gly    #               175    - Asn Thr Pro Leu His Leu Ala Ala Cys Thr As - #n His Val Pro Val Ile    #           190    - Thr Thr Leu Leu Arg Gly Gly Ala Arg Val As - #p Ala Leu Asp Arg Ala    #       205    - Gly Arg Thr Pro Leu His Leu Ala Lys Ser Ly - #s Leu Asn Ile Leu Gln    #   220    - Glu Gly His Ala Gln Cys Leu Glu Ala Val Ar - #g Leu Glu Val Lys Gln    225                 2 - #30                 2 - #35                 2 -    #40    - Ile Ile His Met Leu Arg Glu Tyr Leu Glu Ar - #g Leu Gly Gln His Glu    #               255    - Gln Arg Glu Arg Leu Asp Asp Leu Cys Thr Ar - #g Leu Gln Met Thr Ser    #           270    - Thr Lys Glu Gln Val Asp Glu Val Thr Asp Le - #u Leu Ala Ser Phe Thr    #       285    - Ser Leu Ser Leu Gln Met Gln Ser Met Glu Ly - #s Arg    #   300    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 308 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    - Met Met Leu Gly Arg Phe Arg Glu Ala Leu Gl - #y Asp Ala Gln Gln Ser    #                15    - Val Arg Leu Asp Asp Ser Phe Val Arg Gly Hi - #s Leu Arg Glu Gly Lys    #            30    - Cys His Leu Ser Leu Gly Asn Ala Met Ala Al - #a Cys Arg Ser Phe Gln    #        45    - Arg Ala Leu Glu Leu Asp His Lys Asn Ala Gl - #n Ala Gln Gln Glu Phe    #    60    - Lys Asn Ala Asn Ala Val Met Glu Tyr Glu Ly - #s Ile Ala Glu Thr Asp    #80    - Phe Glu Lys Arg Asp Phe Arg Lys Val Val Ph - #e Cys Met Asp Arg Ala    #                95    - Leu Glu Phe Ala Pro Ala Cys His Arg Phe Ly - #s Ile Leu Lys Ala Glu    #           110    - Cys Leu Ala Met Leu Gly Arg Tyr Pro Glu Al - #a Gln Ser Val Ala Ser    #       125    - Asp Ile Leu Arg Met Asp Ser Thr Asn Ala As - #p Ala Leu Tyr Val Arg    #   140    - Gly Leu Cys Leu Tyr Tyr Glu Asp Cys Ile Gl - #u Lys Ala Val Gln Phe    145                 1 - #50                 1 - #55                 1 -    #60    - Phe Val Gln Ala Leu Arg Met Ala Pro Asp Hi - #s Glu Lys Ala Cys Ile    #               175    - Ala Cys Arg Asn Ala Lys Ala Leu Lys Ala Ly - #s Lys Glu Asp Gly Asn    #           190    - Lys Ala Phe Lys Glu Gly Asn Tyr Lys Leu Al - #a Tyr Glu Leu Tyr Thr    #       205    - Glu Ala Leu Gly Ile Asp Pro Asn Asn Ile Ly - #s Thr Asn Ala Lys Leu    #   220    - Tyr Cys Asn Arg Gly Thr Val Asn Ser Lys Le - #u Arg Lys Leu Asp Asp    225                 2 - #30                 2 - #35                 2 -    #40    - Ala Ile Glu Asp Cys Thr Asn Ala Val Lys Le - #u Asp Asp Thr Tyr Ile    #               255    - Lys Ala Tyr Leu Arg Arg Ala Gln Cys Tyr Me - #t Asp Thr Glu Gln Tyr    #           270    - Glu Glu Ala Val Arg Asp Tyr Glu Lys Val Ty - #r Gln Thr Glu Lys Thr    #       285    - Lys Glu His Lys Gln Leu Leu Lys Asn Ala Gl - #n Leu Lys Phe Arg Asn    #   300    - Tyr Lys Phe Gln    305    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 438 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    - Met Cys Pro Asn Asn Ala Ser Tyr Tyr Gly As - #n Arg Ala Ala Thr Leu    #                15    - Met Met Leu Gly Arg Phe Arg Glu Ala Leu Gl - #y Asp Ala Gln Gln Ser    #            30    - Val Arg Leu Asp Asp Ser Phe Val Arg Gly Hi - #s Leu Arg Glu Gly Lys    #        45    - Cys His Leu Ser Leu Gly Asn Ala Met Ala Al - #a Cys Arg Ser Phe Gln    #    60    - Arg Ala Leu Glu Leu Asp His Lys Asn Ala Gl - #n Ala Gln Gln Glu Phe    #80    - Lys Asn Ala Asn Ala Val Met Glu Tyr Glu Ly - #s Ile Ala Glu Val Asp    #                95    - Phe Glu Lys Arg Asp Phe Arg Lys Val Val Ph - #e Cys Met Asp Arg Ala    #           110    - Leu Glu Phe Ala Pro Ala Cys His Arg Phe Ly - #s Ile Leu Lys Ala Glu    #       125    - Cys Leu Ala Met Leu Gly Arg Tyr Pro Glu Al - #a Gln Phe Val Ala Ser    #   140    - Asp Ile Leu Arg Met Asp Ser Thr Asn Ala As - #p Ala Leu Tyr Val Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Gly Leu Cys Leu Tyr Tyr Glu Asp Cys Ile Gl - #u Lys Ala Val Gln Phe    #               175    - Phe Val Gln Ala Leu Arg Met Ala Pro Asp Hi - #s Glu Lys Ala Cys Val    #           190    - Ala Cys Arg Asn Ala Lys Ala Leu Lys Ala Ly - #s Lys Glu Asp Gly Asn    #       205    - Lys Ala Phe Lys Glu Gly Asn Tyr Lys Leu Al - #a Tyr Glu Leu Tyr Thr    #   220    - Glu Ala Leu Gly Ile Asp Pro Asn Asn Ile Ly - #s Thr Asn Ala Lys Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Tyr Cys Asn Arg Gly Thr Val Asn Ser Lys Le - #u Arg Gln Leu Glu Asp    #               255    - Ala Ile Glu Asp Cys Thr Asn Ala Val Lys Le - #u Asp Asp Thr Tyr Ile    #           270    - Lys Ala Tyr Leu Arg Arg Ala Gln Cys Tyr Me - #t Asp Thr Glu Gln Phe    #       285    - Glu Glu Ala Val Arg Asp Tyr Glu Lys Val Ty - #r Gln Thr Glu Lys Thr    #   300    - Lys Glu His Lys Gln Leu Leu Lys Asn Ala Gl - #n Leu Glu Leu Lys Lys    305                 3 - #10                 3 - #15                 3 -    #20    - Ser Lys Arg Lys Asp Tyr Tyr Lys Ile Leu Gl - #y Val Asp Lys Asn Ala    #               335    - Ser Glu Asp Glu Ile Lys Lys Ala Tyr Arg Ly - #s Arg Ala Leu Met His    #           350    - His Pro Asp Arg His Ser Gly Ala Ser Ala Gl - #u Val Gln Lys Glu Glu    #       365    - Glu Lys Lys Phe Lys Glu Val Gly Glu Ala Ph - #e Thr Ile Leu Ser Asp    #   380    - Pro Lys Lys Lys Thr Arg Tyr Asp Ser Gly Gl - #n Asp Leu Asp Glu Glu    385                 3 - #90                 3 - #95                 4 -    #00    - Gly Met Asn Met Gly Asp Phe Asp Ala Asn As - #n Ile Phe Lys Ala Phe    #               415    - Phe Gly Gly Pro Gly Gly Phe Ser Phe Glu Al - #a Ser Gly Pro Gly Asn    #           430    - Phe Tyr Phe Gln Phe Gly            435    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 245 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    - Met Ala Glu Asp Leu Ser Ala Ala Thr Ser Ty - #r Thr Glu Asp Asp Phe    #                15    - Tyr Cys Pro Val Cys Gln Glu Val Leu Lys Th - #r Pro Val Arg Thr Thr    #            30    - Ala Cys Gln His Val Phe Cys Arg Lys Cys Ph - #e Leu Thr Ala Met Arg    #        45    - Glu Ser Gly Ala His Cys Pro Leu Cys Arg Gl - #y Asn Val Thr Arg Arg    #    60    - Glu Arg Ala Cys Pro Glu Arg Ala Leu Asp Le - #u Glu Asn Ile Met Arg    #80    - Lys Phe Ser Gly Ser Cys Arg Cys Cys Ala Ly - #s Gln Ile Lys Phe Tyr    #                95    - Arg Met Arg His His Tyr Lys Ser Cys Lys Ly - #s Tyr Gln Asp Glu Tyr    #           110    - Gly Val Ser Ser Ile Ile Pro Asn Phe Gln Il - #e Ser Gln Asp Ser Val    #       125    - Gly Asn Ser Asn Arg Ser Glu Thr Ser Thr Se - #r Asp Asn Thr Glu Thr    #   140    - Tyr Gln Glu Asn Thr Ser Ser Ser Gly His Pr - #o Thr Phe Lys Cys Pro    145                 1 - #50                 1 - #55                 1 -    #60    - Leu Cys Gln Glu Ser Asn Phe Thr Arg Gln Ar - #g Leu Leu Asp His Cys    #               175    - Asn Ser Asn His Leu Phe Gln Ile Val Pro Va - #l Thr Cys Pro Ile Cys    #           190    - Val Ser Leu Pro Trp Gly Asp Pro Ser Gln Il - #e Thr Arg Asn Phe Val    #       205    - Ser His Leu Asn Gln Arg His Gln Phe Asp Ty - #r Gly Glu Phe Val Asn    #   220    - Leu Gln Leu Asp Glu Glu Thr Gln Tyr Gln Th - #r Ala Val Glu Glu Phe    225                 2 - #30                 2 - #35                 2 -    #40    - Phe Gln Val Asn Ile                    245    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 245 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    - Met Ser Glu Glu Leu Ser Ala Ala Thr Ser Ty - #r Thr Glu Asp Asp Phe    #                15    - Tyr Cys Pro Val Cys Gln Glu Val Leu Lys Th - #r Pro Val Arg Thr Ala    #            30    - Ala Cys Gln His Val Phe Cys Arg Lys Cys Ph - #e Leu Thr Ala Met Arg    #        45    - Glu Ser Gly Ile His Cys Pro Leu Cys Arg Gl - #y Ser Val Thr Arg Arg    #    60    - Glu Arg Ala Cys Pro Glu Arg Ala Leu Asp Le - #u Glu Asn Ile Met Arg    #80    - Arg Phe Ser Gly Ser Cys Arg Cys Cys Ser Ly - #s Lys Ile Lys Phe Tyr    #                95    - Arg Met Arg His His Tyr Lys Ser Cys Lys Ly - #s Tyr Gln Asp Glu Tyr    #           110    - Gly Val Ser Ser Val Ile Pro Asn Phe Lys Il - #e Ser Gln Asp Ser Val    #       125    - Arg Ser Ser Asn Arg Ser Glu Thr Ser Ala Se - #r Asp Asn Thr Glu Thr    #   140    - Tyr Gln Glu Asp Thr Ser Ser Ser Gly His Pr - #o Thr Phe Lys Cys Pro    145                 1 - #50                 1 - #55                 1 -    #60    - Leu Cys Gln Glu Ser Asn Phe Thr Arg Gln Ar - #g Leu Leu Asp His Cys    #               175    - Asn Ser Asn His Leu Phe Gln Ile Val Pro Va - #l Thr Cys Pro Ile Cys    #           190    - Val Ser Leu Pro Trp Gly Asp Pro Ser Gln Il - #e Thr Arg Asn Phe Val    #       205    - Ser His Leu Asn Gln Arg His Gln Phe Asp Ty - #r Gly Glu Phe Val Asn    #   220    - Leu Gln Leu Asp Glu Glu Thr Gln Tyr Gln Th - #r Ala Val Glu Glu Ser    225                 2 - #30                 2 - #35                 2 -    #40    - Phe Gln Val Asn Met                    245    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of:(a) a nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID NO:1 or a complement thereof;(b) a nucleic acid molecule comprising the nucleotide sequence set forthin SEQ ID NO:2 or a complement thereof; (c) a nucleic acid moleculecomprising the nucleotide sequence set forth in SEQ ID NO:3 or acomplement thereof; (d) a nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID NO:4 or a complement thereof;(e) a nucleic acid molecule comprising the nucleotide sequence set forthin SEQ ID NO:5 or a complement thereof; and (f) a nucleic acid moleculecomprising the nucleotide sequence set forth in SEQ ID NO:6 or acomplement thereof.
 2. An isolated nucleic acid molecule selected fromthe group consisting of:(a) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:31or a complement thereof; (b) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:32or a complement thereof; (c) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:33or a complement thereof; (d) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:34or a complement thereof; (e) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:35or a complement thereof; and (f) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:36or a complement thereof.
 3. An isolated nucleic acid molecule selectedfrom the group consisting of:(a) a nucleic acid molecule comprising thenucleotide sequence contained in the plasmid deposited with ATCC® asAccession Number 98125 or a complement thereof; (b) a nucleic acidmolecule comprising the nucleotide sequence contained in the plasmiddeposited with ATCC® as Accession Number 98126 or a complement thereof;(c) a nucleic acid molecule comprising the nucleotide sequence containedin the plasmid deposited with ATCC® as Accession Number 98127 or acomplement thereof; and (d) a nucleic acid molecule comprising thenucleotide sequence contained in the plasmid deposited with ATCC® asAccession Number 98128 or a complement thereof.
 4. The isolated nucleicacid molecule of any one of claims 1, 2, or 3 which is genomic DNA. 5.The isolated nucleic acid molecule of any one of claims 1, 2, or 3 whichis cDNA.
 6. The isolated nucleic acid molecule of any one of claims 1,2, or 3 which is RNA.
 7. An isolated nucleic acid molecule comprisingthe nucleic acid molecule of any one of claims 1, 2, or 3, and anucleotide sequence encoding a heterologous polypeptide.
 8. An isolatednucleic acid molecule comprising the nucleic acid molecule of any one ofclaims 1, 2, or 3, and a label attached thereto.
 9. A vector comprisingthe nucleic acid molecule of any one of claims 1, 2, or
 3. 10. Thevector of claim 9, which is an expression vector.
 11. A host celltransfected with the vector of claim
 9. 12. A host cell transfected withthe expression vector of claim
 10. 13. A method of producing apolypeptide comprising culturing the host cell of claim 12 in anappropriate culture medium to, thereby, produce the polypeptide.
 14. Anisolated nucleic acid molecule selected from the group consisting of:(a)a nucleic acid molecule consisting of the nucleotide sequence set forthin SEQ ID NO: 1 or a complement thereof; (b) a nucleic acid moleculeconsisting of the nucleotide sequence set forth in SEQ ID NO:2 or acomplement thereof; (c) a nucleic acid molecule consisting of thenucleotide sequence set forth in SEQ ID NO:3 or a complement thereof;(d) a nucleic acid molecule consisting of the nucleotide sequence setforth in SEQ ID NO:4 or a complement thereof; (e) a nucleic acidmolecule consisting of the nucleotide sequence set forth in SEQ ID NO:5or a complement thereof; and (f) a nucleic acid molecule consisting ofthe nucleotide sequence set forth in SEQ ID NO:6 or a complementthereof.
 15. An isolated nucleic acid molecule selected from the groupconsisting of:(a) a nucleic acid molecule which encodes a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO:31 or acomplement thereof; (b) a nucleic acid molecule which encodes apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO:32 or a complement thereof; (c) a nucleic acid molecule which encodesa polypeptide consisting of the amino acid sequence set forth in SEQ IDNO:33 or a complement thereof; (d) a nucleic acid molecule which encodesa polypeptide consisting of the amino acid sequence set forth in SEQ IDNO:34 or a complement thereof; (e) a nucleic acid molecule which encodesa polypeptide consisting of the amino acid sequence set forth in SEQ IDNO:35 or a complement thereof; and (f) a nucleic acid molecule whichencodes a polypeptide consisting of the amino acid sequence set forth inSEQ ID NO:36 or a complement thereof.
 16. An isolated nucleic acidmolecule selected from the group consisting of:(a) a nucleic acidmolecule consisting of the nucleotide sequence contained in the plasmiddeposited with ATCC® as Accession Number 98125 or a complement thereof;(b) a nucleic acid molecule consisting of the nucleotide sequencecontained in the plasmid deposited with ATCC® as Accession Number 98126or a complement thereof; (c) a nucleic acid molecule consisting of thenucleotide sequence contained in the plasmid deposited with ATCC® asAccession Number 98127 or a complement thereof; and (d) a nucleic acidmolecule consisting of the nucleotide sequence contained in the plasmiddeposited with ATCC® as Accession Number 98128 or a complement thereof.17. An isolated nucleic acid molecule comprising 50 consecutivenucleotides of the nucleotide sequence set forth in SEQ ID NO:1, 2, 4,5, or 6, or a complement thereof.
 18. The isolated nucleic acid moleculeof claim 17, comprising 100 consecutive nucleotides of the nucleoridesequence set forth in SEQ ID NO:1, 2, 4, 5, or 6, or a complementthereof.
 19. The isolated nucleic acid molecule of claim 17, comprising150 consecutive nucleotides of the nucleotide sequence set forth in SEQID NO:1, 2, 4, 5, or 6, or a complement thereof.
 20. The isolatednucleic acid molecule of claim 17, comprising 200 consecutivenucleotides of the nucleotide sequence set forth in SEQ ID NO:1, 2, 4,5, or 6, or a complement thereof.
 21. An isolated nucleic acid moleculeconsisting of 50 consecutive nucleorides of the nucleotide sequence setforth in SEQ ID NO:1, 2, 4, 5, or 6, or a complement thereof.
 22. Anisolated nucleic acid molecule comprising 600 consecutive nucleotides ofthe nucleotide sequence set forth in SEQ ID NO:3, or a complementthereof.
 23. An isolated nucleic acid molecule consisting of 600consecutive nucleotides of the nucleotide sequence set forth in SEQ IDNO:3, or a complement thereof.